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WO2020036445A1 - Animal transgénique et embryon transgénique produisant une nucléase artificielle - Google Patents

Animal transgénique et embryon transgénique produisant une nucléase artificielle Download PDF

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Publication number
WO2020036445A1
WO2020036445A1 PCT/KR2019/010388 KR2019010388W WO2020036445A1 WO 2020036445 A1 WO2020036445 A1 WO 2020036445A1 KR 2019010388 W KR2019010388 W KR 2019010388W WO 2020036445 A1 WO2020036445 A1 WO 2020036445A1
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WIPO (PCT)
Prior art keywords
toolbox
cell
genome
animal
transgenic
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/KR2019/010388
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English (en)
Korean (ko)
Inventor
장구
염수영
김경민
이원유
박지현
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Lart Bio Co Ltd
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Lart Bio Co Ltd
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Priority claimed from KR1020190065613A external-priority patent/KR102103103B1/ko
Priority to BR112020000388-3A priority Critical patent/BR112020000388B1/pt
Priority to AU2019275586A priority patent/AU2019275586B2/en
Priority to US16/626,227 priority patent/US12029204B2/en
Priority to JP2019562368A priority patent/JP7060256B2/ja
Application filed by Lart Bio Co Ltd filed Critical Lart Bio Co Ltd
Publication of WO2020036445A1 publication Critical patent/WO2020036445A1/fr
Anticipated expiration legal-status Critical
Priority to AU2021232801A priority patent/AU2021232801B2/en
Priority to AU2021232802A priority patent/AU2021232802B2/en
Priority to JP2022063839A priority patent/JP7430414B2/ja
Priority to JP2024008570A priority patent/JP7671946B2/ja
Priority to US18/593,618 priority patent/US20240298617A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • transgenic animal and a transgenic embryo that produce components of artificial nucleases.
  • the content disclosed herein relates to chimeric transgenic animals and chimeric transgenic embryos having a genome comprising a polynucleotide encoding a component of an artificial nuclease.
  • the content disclosed by the present specification relates to a polynucleotide encoding a component of an artificial nuclease and a first cell having a genome in which the polynucleotide encoding a component of an artificial nuclease is located at a first locus.
  • a transgenic embryo and a transgenic animal comprising a second cell having a genome located at a second locus different from the first locus.
  • Also disclosed herein is a first cell having a genome in which a first polynucleotide encoding a component of an artificial nuclease and a second encoding an component of an artificial nuclease is located.
  • a transgenic embryo and a transgenic animal comprising a second cell having a genome whose polynucleotide is located at a first locus. At this time, the sequence of the first polynucleotide and the second polynucleotide is different.
  • Gene manipulation using gene editing tools including Crisper / Kas9, is being applied to animals and plants of various species.
  • the genetic manipulation that has been generally performed has been used a method of providing a gene editing tool to a cell or an individual from the outside, there is a problem that the efficiency of genetic manipulation is low when the gene editing tool is injected into the cell or individual from the outside.
  • the gene editing tool may be expressed in the animal or cell, resulting in higher genetic engineering efficiency.
  • transgenic animal having a gene inserted into a gene encoding a gene editing tool it is possible to produce a large number of animals knocked out or knocked out of various genes. This will allow transgenic animals with a genome inserted into a gene encoding a gene editing tool to be used as a platform technology.
  • transgenic animal when the transgenic animal is a large animal, its utility may be better than that of the small animal.
  • One subject disclosed by the present application relates to a transgenic animal comprising a polynucleotide encoding a component of an artificial nuclease.
  • transgenic embryos comprising polynucleotides encoding components of an artificial nuclease.
  • the problem to be solved by the present application is to enable more efficient genetic manipulation using the transgenic animal or the transgenic embryo in which the components of the artificial nuclease can be expressed.
  • transgenic animal or the transgenic embryo in which a component of an artificial nuclease can be expressed, a plurality of cells, embryos and / or knocked out or knocked out of various genes. It's about making animals work.
  • a first cell having a genome comprising a first toolbox; And a second cell having a genome comprising a second toolbox.
  • the first and second toolboxes comprise at least one or more of a polynucleotide encoding an RNA-guide endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, wherein Wherein the target site is an endo-polynucleotide of the animal or is an exo-polynucleotide contained on the animal's genome, located between a first ITR sequence and a second ITR sequence,
  • the first toolbox is at a first locus in the genome of the first cell
  • the second toolbox is at a second locus in the genome of the second cell
  • the first locus and the second locus are provided with different transgenic animals.
  • a first cell having a genome comprising a first toolbox; And a second cell having a genome comprising a second toolbox.
  • the first and second toolboxes comprise at least one or more of a polynucleotide encoding an RNA-guide endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, wherein wherein the target site is an endo-polynucleotide of the animal or is an exo-polynucleotide contained on the animal's genome, located between a first ITR sequence and a second ITR sequence-
  • the sequence of the first toolbox and the sequence of the second toolbox are different.
  • the first toolbox is at a first locus in the genome of the first cell
  • the second toolbox is at a second locus in the genome of the second cell
  • the first locus and the second locus are provided with the same transgenic animal.
  • a first cell having a genome comprising a first toolbox; And a second cell having a genome comprising a second toolbox.
  • the first and second toolboxes comprise at least one or more of a polynucleotide encoding an RNA-guide endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, wherein Wherein the target site is an endo-polynucleotide of the embryo or is an exo-polynucleotide contained on the embryo's genome, located between a first ITR sequence and a second ITR sequence,
  • the first toolbox is at a first locus in the genome of the first cell
  • the second toolbox is at a second locus in the genome of the second cell
  • the first locus and the second locus are provided with different transgenic embryos.
  • a first cell having a genome comprising a first toolbox; And a second cell having a genome comprising a second toolbox.
  • the first and second toolboxes comprise at least one or more of a polynucleotide encoding an RNA-guide endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site, wherein Wherein the target site is an endo-polynucleotide of the embryo or is an exo-polynucleotide contained on the embryo's genome, located between a first ITR sequence and a second ITR sequence
  • the sequence of the first toolbox and the sequence of the second toolbox are different.
  • the first toolbox is at a first locus in the genome of the first cell
  • the second toolbox is at a second locus in the genome of the second cell
  • the first locus and the second locus are provided with the same transgenic embryo.
  • a first cell having a genome comprising a first toolbox and a target site; And a second cell having a genome comprising a second toolbox and a modified site.
  • the first toolbox and the second toolbox comprise one or more of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to the target site,
  • the target site comprises a first region, a second region and a third region, wherein the first region is located at a 5 'end of the second region, and the third region is a 3' of the second region Located at the end-,
  • the deformed site comprises a fourth region, a fifth region and a sixth region, wherein the fourth region is located at the 5 'end of the fifth region, and the sixth region is 3 of the fifth region 'Located at the end-,
  • the sequence of the first region and the sequence of the fourth region are identical to each other,
  • Transgenic animals are provided that differ in sequence from the second region and from the sequence from the fifth region.
  • a first cell having a genome comprising a first toolbox and a target site; And a second cell having a genome comprising a second toolbox and a modified site.
  • the first toolbox and the second toolbox comprise one or more of a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid capable of specifically binding to the target site,
  • the target site comprises a first region, a second region and a third region, wherein the first region is located at a 5 'end of the second region, and the third region is a 3' of the second region Located at the end-,
  • the deformed site comprises a fourth region, a fifth region and a sixth region, wherein the fourth region is located at the 5 'end of the fifth region, and the sixth region is 3 of the fifth region 'Located at the end-,
  • the sequence of the first region and the sequence of the fourth region are identical to each other,
  • sequence of the second region and the sequence of the fifth region are provided with a transgenic embryo that is different from each other.
  • a method for producing a transformed embryo in which a component of an artificial nuclease is expressed the vector comprising a transposon gene and a polynucleotide encoding a component of the artificial nuclease Microinjecting the fertilized egg or embryo, wherein the vector is a plasmid vector or a viral vector;
  • the vector is a plasmid vector or a viral vector;
  • a method for producing a transformed embryo in which a component of an artificial nuclease is expressed i) preparing a transformed donor cell in which the component of the artificial nuclease is expressed that;
  • a method for producing a transgenic animal in which a component of an artificial nuclease is expressed i) producing a transgenic embryo in which the component of the artificial nuclease is expressed that; And ii) transplanting the transgenic embryo into the uterus of the surrogate mother.
  • An embryo manufacturing method having a genetically edited genome comprising a is provided.
  • transgenic donor cell comprising a polynucleotide encoding an RNA-guided endonuclease, said gene having a genetically edited genome at said target site; And ii) transplanting the nucleus of the transgenic donor cell into a denuclearized egg.
  • an embryo comprising a polynucleotide encoding an RNA-guided endonuclease and having a genetically edited genome at said target site; And ii) transplanting the embryo into a surrogate mother; An animal production method having a genetically edited genome comprising a is provided.
  • An embryo manufacturing method having a genetically edited genome comprising a is provided.
  • a manufacturing method having a genetically edited genome at a target site present in the genome
  • a transgenic animal comprising a polynucleotide encoding a component of an artificial nuclease.
  • a transgenic animal capable of more efficiently genetic manipulation can be provided.
  • platform transgenic animals can be provided for the production of cells, embryos and / or animals in which various genes are knocked in or knocked out.
  • a transgenic embryo can be provided comprising a polynucleotide encoding a component of an artificial nuclease.
  • a transgenic embryo capable of more efficiently genetic manipulation can be provided.
  • platform transgenic embryos can be provided for the production of cells, embryos and / or animals in which various genes have been knocked in or knocked out.
  • FIG. 1 shows a toolbox comprising a polynucleotide encoding a component of an artificial nuclease.
  • FIG. 2 shows some examples of polynucleotides encoding components of artificial nucleases.
  • 3 shows chromosomes formed from genomes present in cells.
  • FIG. 4 shows some forms in which the toolbox can be inserted on a chromosome.
  • Figure 6 shows a one-celled embryo having a genome inserted with a toolbox and two-celled embryos, four-celled embryos, eight-celled embryos and 16-celled embryos divided from the one-celled embryos.
  • FIG. 7 shows two-cell embryos, four-cell embryos, eight-cell embryos, and 16-cell embryos in which the toolbox is inserted only in the genome of some cells.
  • a toolbox including a polynucleotide encoding an RNA-guide endonuclease and a polynucleotide encoding a guide nucleic acid and a specific target site in the genome into which the toolbox is inserted does not include an expression control element. Indicates.
  • FIG. 11 shows some examples of toolboxes incorporating polynucleotides having a PAM sequence.
  • FIG. 12 shows one process of genetic editing within a toolbox containing polynucleotides having a PAM sequence.
  • FIG. 13 shows one process that is genetically edited in a toolbox containing a polynucleotide having a PAM sequence.
  • FIG. 14 shows one process of gene editing in a toolbox containing a polynucleotide having a PAM sequence.
  • FIG. 15 shows a process for knocking out a polynucleotide encoding a target protein and a polynucleotide encoding a linker to a donor polynucleotide, and the target protein is produced from a cell in which the donor polynucleotide is knocked or a transgenic animal comprising the cell. Indicate the process.
  • Figure 16 illustrates one method of selecting cells having a genome inserted with a surface toolbox.
  • 17 shows one method of selecting for cells in which gene editing has occurred in a surface toolbox.
  • FIG. 18 shows the genome into which the suicide toolbox is inserted, the cell with the genome into which the suicide toolbox is inserted, the form in which gene editing has been performed in the suicide toolbox, and the cell in which the gene editing has been performed in the suicide toolbox.
  • FIG. 20 shows a process of distinguishing an embryo having an XX chromosome and an embryo having an XY chromosome by the genome into which the XY chromosome division toolbox is inserted and the XY chromosome division toolbox.
  • FIG. 21 shows an example of a removal toolbox in which the expression of transposase can be regulated by an induction promoter and a tissue specific promoter.
  • FIG. 22 shows an example of a removal toolbox in which the expression of transposase can be modulated by an induction promoter and a tissue specific promoter.
  • FIG. 23 shows an example of a removal toolbox in which expression of recombinase can be regulated by an induction promoter and a tissue specific promoter.
  • FIG. 24 shows a portion of a vector containing a gene encoding a green fluorescent protein (green fluorescent protein gene) and a portion of a vector containing a gene encoding a red fluorescent protein (red fluorescent protein gene).
  • FIG. 25 shows fluorescent protein expression in donor cells having a genome inserted with a toolbox containing a gene encoding a fluorescent protein (fluorescent protein gene).
  • FIG. 26 shows RT-PCR showing whether the green fluorescent protein gene or the red fluorescent protein gene is expressed in cloned embryos, and DNA PCR results confirming that the green fluorescent protein gene or the red fluorescent protein gene is inserted into the genome of the cloned embryo. Indicates.
  • FIG. 27 shows expression of fluorescent proteins in cloned embryos with a genome inserted with a toolbox containing fluorescent protein genes.
  • Figure 29 shows the results of confirming the expression of the yellow fluorescent protein in a transgenic cow having a genome inserted with a toolbox containing a gene encoding a yellow fluorescent protein (yellow fluorescent protein gene).
  • Figure 30 shows the results of confirming the expression of the green fluorescent protein in a transgenic cow having a genome inserted with a toolbox containing the green fluorescent protein gene.
  • FIG. 31 shows a transformed cow having a genome inserted with a toolbox including an expression control element, a green fluorescent protein gene and a red fluorescent protein gene, and a red fluorescent protein when a substance affecting the expression control element is treated in the transformed cow. The result of confirming the expression of is shown.
  • DNA PCR and RT-PCR results confirming the insertion and transcription of green fluorescent protein genes in a transgenic cow having a genome inserted with a toolbox containing an expression regulatory element, a green fluorescent protein gene, and a red fluorescent protein gene
  • DNA PCR results confirm that the green fluorescent protein gene is removed and only the red fluorescent protein gene is present as a result of the treatment of substances affecting the expression regulatory elements.
  • FIG. 33 shows fluorescent protein expression in progeny cattle and progeny bovine primary cells with a genome inserted with a toolbox containing fluorescent protein genes.
  • Fig. 34 shows the results of DNA PCR analysis confirming the presence of fluorescent protein genes in the genome of progeny cows with a genome into which a toolbox containing fluorescent protein genes is inserted.
  • 35 shows the difference in the degree of fluorescent protein expression according to the number of toolboxes containing the green fluorescent protein gene inserted on the genome.
  • 36 shows visual and ELISA results confirming the expression of green fluorescent protein in the milk of a cow having a genome inserted with a toolbox including the green fluorescent protein gene.
  • FIG. 37 shows sgRNA expression vectors and Cas9 expression vectors for knocking out green fluorescent protein genes inserted into the genome of cells.
  • FIG. 39 shows donor DNA when gRNA, Cas9, and donor DNA (puromycin resistance gene) targeting green fluorescent protein gene are transfected to primary cells having a genome into which the toolbox containing the green fluorescent protein gene is inserted. Shows a result showing that K is ok.
  • green fluorescent protein is not expressed in primary cells transfected with sgRNA expression vector, crisper / cas 9 expression vector and donor DNA (puromycin resistance gene) expression vector targeting green fluorescent protein.
  • FIG. 41 shows the process by which donor polynucleotides are knocked down by targeting the green fluorescent protein gene present in the genome of bovine primary cells.
  • FIG. 42 shows that red fluorescent protein is expressed after red fluorescent protein gene is knocked down by targeting green fluorescent protein gene in a bovine primary cell having a green fluorescent protein gene inserted therein.
  • FIG. 43 shows a transformed embryo and a transgenic cow having a vector containing a toolbox containing the spCas9 gene, a genome into which a toolbox containing the spCas9 gene is inserted.
  • FIG. 44 shows visual results showing the expression of red fluorescent protein in primary cells of a transgenic cow having a genome inserted with a toolbox containing spCas9 gene, DNA PCR results showing the insertion of spCas9 gene into the genome of the transgenic cow, and the transformation RT-PCR results show the expression of the spCas9 gene inserted into the bovine genome.
  • 45 illustrates a process of knocking out a target gene using a cow having a genome inserted with a Cas9 gene.
  • FIG. 46 shows knockout results of a PRNP gene, a Beta-lactoglobulin (BLG) gene, a Retinoblastoma 1 (Rb1) gene, a Nanog gene, a P53 gene, and a beta-casein (BCN) gene using a cow having a genome inserted with the spCas9 gene. Indicates.
  • FIG. 48 shows that red fluorescent protein is expressed in primary cells of progeny cattle obtained by spontaneous crossbreeding of cows with spCas9 gene inserted and spCas9 gene and Fat1 gene are inserted into genome of the progeny cattle by DNA PCR.
  • Figure 49 shows the results of electrophoresis confirming the indel of the PRNP gene after transfection of the sgRNA targeting the PRNP gene to the primary cell of the transgenic cow having the genome inserted spCas9 gene.
  • 50 shows a portion of donor polynucleotide vector for HDR and a portion of donor polynucleotide vector for HITI.
  • Fig. 51 shows the results of confirming knock-in of the mcherry gene by HITI.
  • Figure 53 shows mcherry expression in the mcherry gene knocked embryos produced through somatic cell nuclear transfer and mcherry expression in the embryo.
  • 54 shows part of the final expression vector for expressing spCas9 and sgRNA.
  • FIG. 55 shows the expression of red fluorescent protein in primary cells of transgenic cattle with a toolbox containing the spCas9 gene and a polynucleotide encoding sgRNA and the indel of the beta-lactoglobulin gene in fibroblasts of the transgenic cattle fPCR results are shown.
  • RNA-guide endonucleases 56 shows a portion of a vector in which the expression of RNA-guide endonucleases can be regulated.
  • Figure 57 shows the results of DNA PCR confirmed whether the target gene according to the presence or absence of Cre recombinase treatment in cells that can control the expression of RNA-guided endonuclease.
  • FIG. 58 is a sequence of a donor vector for HDR, in which a highlighted part of the entire sequence shows the first homologous sequence and the second homologous sequence.
  • a transgenic embryo may be provided having a genome into which a polynucleotide encoding a component of an artificial nuclease is inserted.
  • a transgenic fertilized egg or transgenic embryo having a genome comprising a polynucleotide encoding an RNA-guide endonuclease comprised between a first ITR sequence and a second ITR sequence.
  • the embryo may be an embryo of the ungulate.
  • a transgenic fertilized egg or embryo further comprising a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site present in the fertilized egg or embryo genome between the first ITR sequence and the second ITR sequence. May be provided.
  • At least one of the 5 'end of the polynucleotide encoding the RNA-guide endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid may include an expression control element.
  • a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, wherein the first toolbox is present at the first locus and is A second toolbox is present at the second locus, wherein the first locus and the second locus may be provided with different transgenic embryos.
  • the first toolbox and the second toolbox may include at least one or more of a polynucleotide encoding an RNA-guide endonnucleotide and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site.
  • the target site may be an endo-polynucleotide of the embryo or an exo-polynucleotide contained on the embryo's genome, which may be included between the first and second ITR sequences.
  • the sequence of the first toolbox and the sequence of the second toolbox may be the same or different.
  • a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, wherein the first toolbox is present at a first locus
  • the second toolbox is in a second locus, and the first locus and the second locus may be provided with the same transgenic embryo.
  • the first toolbox and the second toolbox may include at least one or more of a polynucleotide encoding an RNA-guide endonnucleotide and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site.
  • the target site may be an endo-polynucleotide of the embryo or an exo-polynucleotide contained on the embryo's genome, which may be included between the first and second ITR sequences.
  • the sequence of the first toolbox and the sequence of the second toolbox are different.
  • the genome of the first cell may further comprise a third toolbox identical to the sequence of the first toolbox.
  • the genome of the second cell may further comprise a fourth toolbox identical to the sequence of the second toolbox.
  • the transgenic embryo may further comprise a third cell having a genome that does not comprise a polynucleotide encoding the RNA-guide endonuclease and the guide nucleic acid.
  • a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a target site and a second cell having a genome comprising the target site.
  • the first toolbox comprises at least one or more of a polynucleotide encoding a first RNA-guided endonuclease and a polynucleotide encoding a first guide nucleic acid capable of binding to the target site, wherein the second cell
  • the genome of may not comprise a polynucleotide encoding a second RNA-guided endonuclease and a polynucleotide encoding a second guide nucleic acid capable of specifically binding to said target site.
  • the 5 'end and the 3' end of the first toolbox may further include an ITR sequence.
  • the sequence of the polynucleotide encoding the first RNA-guided endonuclease and the sequence of the polynucleotide encoding the second RNA-guided endonuclease may be the same or different.
  • the sequence of the polynucleotide encoding the first guide nucleic acid and the sequence of the polynucleotide encoding the second guide nucleic acid may be the same or different.
  • the target site may be an endo-polynucleotide. Specifically, the target site may be a base sequence of 18bp to 25bp present on the genome of the transgenic embryo.
  • the target site may be an exo-polynucleotide.
  • the target site is a sequence adjacent to the 5 'end or 3' end of the PAM sequence, and the target site and the PAM sequence may be included between the first ITR sequence and the second ITR sequence.
  • a method for preparing a transgenic embryo having a genome inserted with a polynucleotide encoding a component of an artificial nuclease can be provided.
  • One method for producing the transgenic embryo may comprise microinjecting a vector comprising a polynucleotide encoding a transposon gene and a component of an artificial nuclease into a fertilized egg or embryo.
  • it may include microinjecting the transposaase that can interact with the transposon gene into the fertilized egg or embryo.
  • the transposase may be in the form of a protein, polypeptide, or polynucleotide encoding the transposase.
  • the polynucleotide may be contained in a plasmid vector or viral vector.
  • the polynucleotides encoding the transposase may be included in one vector and the polynucleotides encoding the transposon gene and the components of the artificial nuclease and microinjected into the fertilized egg or embryo.
  • One type of transgenic embryo that can be produced by the method is a first cell having a genome containing a polynucleotide encoding a component of a first artificial nuclease in a first locus and a second artificial nuclease.
  • the polynucleotide encoding the component may comprise a second cell having a genome contained in a second locus that is different from the first locus.
  • the sequence of the polynucleotides encoding the components of the first artificial nuclease and the polynucleotides encoding the components of the second artificial nuclease may be the same or different.
  • Other forms of transgenic embryos that can be produced by the method include a first cell having a genome containing a polynucleotide encoding a component of a first artificial nuclease at a first locus and a second artificial nuclease.
  • the polynucleotide encoding the component may comprise a second cell having a genome contained in the first locus.
  • the sequence of the polynucleotides encoding the components of the first artificial nuclease and the polynucleotides encoding the components of the second artificial nuclease may be the same or different.
  • Other methods for preparing the transgenic embryos may include preparing a transgenic donor cell in which a component of an artificial nuclease is expressed and implanting the nucleus of the transgenic donor cell into a denuclear egg.
  • Producing the transformed donor cell may comprise transforming the cell with a vector comprising a transposon gene and a polynucleotide encoding a component of the artificial nuclease.
  • preparing the transgenic donor cell may further include transforming the cell with a transposase that can interact with the transposon gene.
  • the transposase may be in the form of a protein, polypeptide, or polynucleotide encoding the transposase.
  • the polynucleotide may be contained in a plasmid vector or viral vector. Furthermore, the polynucleotides encoding the transposase may be included in one vector and the polynucleotides encoding the transposon gene and the components of the artificial nuclease and microinjected into the fertilized egg or embryo.
  • a transgenic animal having a genome inserted with a polynucleotide encoding a component of an artificial nuclease may be provided.
  • a transgenic animal having a genome comprising a polynucleotide encoding an RNA-guide endonuclease comprised between a first ITR sequence and a second ITR sequence.
  • the animal may be a cow worm.
  • a transgenic animal may be provided further comprising a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site present in the animal between the first ITR sequence and the second ITR sequence.
  • At least one of the 5 'end of the polynucleotide encoding the RNA-guide endonuclease and the 5' end of the polynucleotide encoding the guide nucleic acid may include an expression control element.
  • a chimeric transgenic animal comprising a first cell having a genome comprising a toolbox and a second cell having a genome not comprising a toolbox.
  • a transgenic animal may be provided comprising a first cell having a genome comprising a first toolbox and a target site and a second cell having a genome comprising the target site.
  • the first toolbox comprises at least one or more of a polynucleotide encoding a first RNA-guided endonuclease and a polynucleotide encoding a first guide nucleic acid capable of binding to the target site, wherein the second cell
  • the genome of may not comprise a polynucleotide encoding a second RNA-guided endonuclease and a polynucleotide encoding a second guide nucleic acid capable of specifically binding to said target site.
  • the 5 'end and the 3' end of the first toolbox may further include an ITR sequence.
  • the sequence of the polynucleotide encoding the first RNA-guided endonuclease and the sequence of the polynucleotide encoding the second RNA-guided endonuclease may be the same or different,
  • the sequence of the polynucleotide encoding the first guide nucleic acid and the sequence of the polynucleotide encoding the second guide nucleic acid may be the same or different.
  • the target site may be an endo-polynucleotide. Specifically, the target site may be a base sequence of 18bp to 25bp present on the genome of the transgenic animal.
  • the target site may be an exo-polynucleotide.
  • the target site is a sequence adjacent to the 5 'end or 3' end of the PAM sequence, and the target site and the PAM sequence may be included between the first ITR sequence and the second ITR sequence.
  • a transgenic animal comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, the first toolbox being at a first locus
  • the second toolbox is in a second locus, and the first locus and the second locus may be provided with different transgenic animals.
  • the first toolbox and the second toolbox may include at least one or more of a polynucleotide encoding an RNA-guide endonnucleotide and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site.
  • the target site may be an endo-polynucleotide of the animal, or may be included between the first and second ITR sequences as an exo-polynucleotide contained on the genome of the animal.
  • the sequence of the first toolbox and the sequence of the second toolbox may be the same or different.
  • a transgenic animal comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox, the first toolbox being at a first locus
  • the second toolbox is in a second locus, and the first locus and the second locus may be provided with the same transgenic animal.
  • the first toolbox and the second toolbox may include at least one or more of a polynucleotide encoding an RNA-guide endonnucleotide and a polynucleotide encoding a guide nucleic acid capable of specifically binding to a target site.
  • the target site may be an endo-polynucleotide of the animal, or may be included between the first and second ITR sequences as an exo-polynucleotide contained on the genome of the animal.
  • the sequence of the first toolbox and the sequence of the second toolbox are different.
  • the genome of the first cell may further comprise a third toolbox identical to the sequence of the first toolbox.
  • the genome of the second cell may further comprise a fourth toolbox identical to the sequence of the second toolbox.
  • the transgenic animal may further comprise a third cell having a genome that does not include a polynucleotide encoding the RNA-guide endonuclease and the guide nucleic acid.
  • a method of making a transgenic animal having a genome inserted with a polynucleotide encoding a component of an artificial nuclease can be provided.
  • One method for producing the transgenic animal may include microinjecting a vector comprising a transposon gene and a polynucleotide encoding a component of the artificial nuclease into a fertilized egg or embryo.
  • preparing the transgenic embryo may further include micro-injecting the transposease into the fertilized egg or embryo, which may interact with the transposon gene.
  • Other methods for producing the transgenic animal may include preparing a transgenic donor cell in which the component of the artificial nuclease is expressed and transplanting the nucleus of the transgenic donor cell into a denuclear egg of the animal. have.
  • Producing the transformed donor cell may comprise transforming the cell with a vector comprising a transposon gene and a polynucleotide encoding a component of the artificial nuclease.
  • preparing the transgenic donor cell may further include transforming the cell with a transposase that can interact with the transposon gene.
  • the transposase may be in the form of a protein, polypeptide, or polynucleotide encoding the transposase.
  • the polynucleotide may be contained in a plasmid vector or viral vector. Furthermore, the polynucleotides encoding the transposase may be in a form contained in one vector with the polynucleotides encoding the transposon gene and the components of the artificial nuclease.
  • a transgenic embryo having a genetically edited genome can be provided.
  • a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid between a first ITR sequence and a second ITR sequence, and having a genome with the endo-polynucleotide knocked out Transgenic embryos may be provided.
  • the guide nucleic acid may specifically bind to the endo-polynucleotide.
  • the endo-polynucleotide knocked out form may include: i) a form in which at least one nucleotide of the endo-polynucleotide sequence is not included; ii) at least one nucleotide is further included in the endo-polynucleotide sequence. Iii) one or more nucleotides of the endo-polynucleotide sequence is removed, and one or more nucleotides are further included.
  • a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a target site and a second cell having a genome comprising a second toolbox and a modified site.
  • the sequence of the first toolbox and the second toolbox may be the same or different.
  • the target site may be an endo-polynucleotide.
  • the target site may be an exo-polynucleotide.
  • the modified site may be a sequence of the target site changed by gene editing.
  • the target site may include a first region, a second region, and a third region, and the modified site may include a fourth region, a fifth region, and a sixth region.
  • the sequence of the first region and the sequence of the fourth region are identical to each other, the sequence of the third region and the sequence of the sixth region are the same, and the sequence of the second region and the sequence of the fourth region Are different from each other.
  • PAM sequences may be included in the second region and the fifth region.
  • the PAM sequence may be included in the third region and the sixth region.
  • the sequence of the fifth region is i) a form in which at least one nucleotide of the sequence of the second region is not included. ii) one or more nucleotides may be additionally included in the sequence of the second region, or iii) one or more nucleotides of the sequence of the second region may be removed and one or more nucleotides may be additionally included.
  • one or more nucleotides further included may include an editing enabling component, a polynucleotide encoding a protein or RNA, a polynucleotide encoding a non-functional polypeptide, an untranslated RNA It may comprise one or more of a polynucleotide encoding a, a non-transcribed polynucleotide, an artificial intron and expression control elements.
  • a transgenic embryo comprising a first cell having a genome comprising a first toolbox and a target site, and a second cell having a genome that does not include a toolbox but contains a modified gene.
  • the first toolbox may include one or more of a polynucleotide encoding an RNA-guide endonuclease and a polynucleotide encoding a guide nucleic acid.
  • the modified site may be a sequence of the target site changed by gene editing. Specifically, the target site may include a first region, a second region, and a third region. The modified site may include a fourth region, a fifth region, and a sixth region.
  • sequence of the first region and the sequence of the fourth region are identical to each other
  • sequence of the third region and the sequence of the sixth region are the same
  • sequence of the second region and the sequence of the fourth region Are different from each other.
  • the sequence of the fifth region is i) a form in which at least one nucleotide of the sequence of the second region is not included. ii) one or more nucleotides may be additionally included in the sequence of the second region, or iii) one or more nucleotides of the sequence of the second region may be removed and one or more nucleotides may be additionally included.
  • a transgenic animal having a genetically edited genome can be provided.
  • a polynucleotide encoding an RNA-guided endonuclease and a polynucleotide encoding a guide nucleic acid between a first ITR sequence and a second ITR sequence, and having a genome with the endo-polynucleotide knocked out Transgenic animals may be provided.
  • the guide nucleic acid may specifically bind to the endo-polynucleotide.
  • the endo-polynucleotide knocked out form may include: i) a form in which at least one nucleotide of the endo-polynucleotide sequence is not included; ii) at least one nucleotide is further included in the endo-polynucleotide sequence. Iii) one or more nucleotides of the endo-polynucleotide sequence is removed, and one or more nucleotides are further included.
  • a transgenic animal comprising a first cell having a genome comprising a first toolbox and a target site and a second cell having a genome comprising a second toolbox and a modified site.
  • the sequence of the first toolbox and the second toolbox may be the same or different.
  • the first toolbox and / or the second toolbox may include one or more of a polynucleotide encoding an RNA-guide endonuclease and a polynucleotide encoding a guide nucleic acid.
  • the target site may be an endo-polynucleotide.
  • the target site may be an exo-polynucleotide.
  • the modified site may be a sequence of the target site changed by gene editing.
  • the target sequence may include a first region, a second region, and a third region
  • the modified sequence may include a fourth region, a fifth region, and a sixth region.
  • the sequence of the first region and the sequence of the fourth region are identical to each other
  • the sequence of the third region and the sequence of the sixth region are the same
  • the sequence of the second region and the sequence of the fourth region Are different from each other.
  • PAM sequences may be included in the second region and the fifth region.
  • the PAM sequence may be included in the third region and the sixth region.
  • the sequence of the fifth region is i) a form in which at least one nucleotide of the sequence of the second region is not included.
  • one or more nucleotides may be additionally included in the sequence of the second region, or iii) one or more nucleotides of the sequence of the second region may be removed and one or more nucleotides may be additionally included.
  • one or more nucleotides further included may include an editing enabling component, a polynucleotide encoding a protein or RNA, a polynucleotide encoding a non-functional polypeptide, an untranslated RNA It may comprise one or more of a polynucleotide encoding a, a non-transcribed polynucleotide, an artificial intron and expression control elements.
  • the target site and the modified site may be sequences adjacent to a PAM sequence.
  • the first ITR sequence may be included in the 5 'direction of the target site and the modified site, and the second ITR sequence may be included in the 3' direction.
  • the term 'transformation' of the present application refers to artificially modifying a polynucleotide included in an animal genome of a cell.
  • the modification includes removing a part of a polynucleotide included in an animal genome in a cell, substituting a part, and a nucleotide or polynucleotide in an animal genome. It includes inserting.
  • the term 'transformation' of the present application includes adding, altering, or removing proteins or RNA that can be expressed in a cell.
  • RNA-guided endonuclease is a base on the animal genome capable of complementarily binding to a portion of the guide nucleic acid on the animal genome. Recognition of sequences can result in site-specific trandformation.
  • the term 'transformed cell' of the present application refers to a cell containing the transformed portion of the animal genome in the cell.
  • the first cell comprising the transformed portion of the intracellular animal genome can divide.
  • the animal genome of the second cell obtained through cell division of the first cell may include a part having the same base sequence as the transformed part of the first cell.
  • the term 'transformed cell' of the present application includes the first cell and the second cell.
  • the term 'transformed animal' in the present application means an animal including at least one transformed cell.
  • Animal F0 may comprise a first transformed cell.
  • the animal F0 can produce offspring F1.
  • the one or more cells included in the F1 and F1 or less progeny may include an animal genome including a part having the same base sequence as the transformed part of the animal genome in the first transformed cell. Can be.
  • the term 'transgenic animal' of the present application includes the above F0, F1, and F1 or less offspring.
  • the animal can be referred to as a transgenic animal if the animal F1 contains transformed cells.
  • F1 can be said to be a transgenic animal.
  • 'endo-polynucleotide' of the present application refers to a polynucleotide included in an animal genome of a cell in which no transformation occurs.
  • the first cell that has not undergone transformation can undergo cell division.
  • the second cell obtained through cell division of the first cell may be a transformed cell.
  • the second cell may include a polynucleotide having the same sequence as the endo-polynucleotide included in the first cell.
  • the term 'endo-polynucleotide' of the present application includes a polynucleotide having the same sequence as an endo-polynucleotide of a first cell among polynucleotides included in the second cell.
  • exo-polynucleotide' of the present application means a polynucleotide that is introduced to a cell.
  • the cell may be a transformed cell. Or may not be a transformed cell.
  • the exo-polynucleotide may be inserted into the animal genome in the cell or exist independently of the animal genome in the cell.
  • the first cell into which the exo-polynucleotide is introduced may divide into cells.
  • the second cell obtained through cell division of the first cell may include a polynucleotide having the same sequence as the exo-polynucleotide.
  • the term 'exo-polynucleotide' of the present application includes a polynucleotide having the same sequence as an exo-polynucleotide of a first cell among polynucleotides included in the second cell. .
  • the F1 and F1 or less progeny may include a cell including a polynucleotide having the same sequence as the exo-polynucleotide of the F0.
  • the term 'exo-polynucleotide' of the present application refers to a polynucleotide having the same sequence as the exo-polynucleotide of F0 among polynucleotides contained in cells of the F1 and F1 or less progeny. Include.
  • 'insertion' in the present application includes 'nucleotide insertion' and 'polynucleotide insertion'.
  • the term 'nucleotide insertion' refers to adding a nucleotide to an intermediate, 5 'end or 3' end of a nucleic acid.
  • the term 'polynucleotide insertion' refers to adding polynucleotide to the middle, 5 'end or 3' end of a nucleic acid.
  • 'deletion' in the present application includes 'nucleotide deletion' and 'polynucleotide deletion'.
  • the term 'nucleotide deletion' means to remove nucleotides contained in a nucleic acid.
  • 'polynucleotide deletion' refers to the removal of polynucleotides contained in a nucleic acid.
  • substitution in the present application includes 'nucleotide substitution' and 'polynucleotide substitution'.
  • 'nucleotide substitution' refers to replacing a nucleotide included in a nucleic acid with another nucleotide.
  • 'polynucleotide substitution' means to change a polynucleotide included in a nucleic acid to another polynucleotide.
  • knock-in' in the present application means insertion or substitution of an exo-polynucleotide comprising a gene onto an animal genome in a cell.
  • exo-polynucleotides comprising a human albumin gene can be inserted onto a non-human animal genome in a cell.
  • the cell containing the non-human animal genome can express human albumin. This can be called the human albumin gene knockin.
  • knockout' in the present application means that a gene present on an animal genome in a cell cannot function.
  • a polynucleotide in which a gene is located on an animal genome in a cell may be transformed to knock out the gene.
  • an exo-polynucleotide comprising a human albumin gene is placed on a non-human animal genome in a cell. It can be inserted into the base sequence corresponding to the exon of the human albumin gene. In this case, the cells are unable to express non-human albumin. This can be referred to as a non-human albumin gene knockout.
  • an artificial nuclease that targets an exon portion of a non-human albumin gene present on a non-human animal genome in a cell as a target site.
  • the exon portion may be removed using
  • the cells are unable to express non-human albumin. This can be referred to as a non-human albumin gene knockout.
  • engineered nuclease' in the present application means a protein or complex comprising a protein capable of site-specific transfoemation in an animal genome. do.
  • the protein may be a non-modified protein or a modified / engineered protein found in nature.
  • ZFN zinc finger nuclease
  • TALEN Transcription Activator-Like Effector Nuclease
  • CRISPR / enzyme system CRISPR / enzyme system. It may include, but is not limited thereto.
  • the target site of an artificial nuclease may refer to a region in a nucleic acid that one component of an artificial nuclease can recognize.
  • the target site of the CRISPR / enzyme system may be one or more base sequences capable of binding identically or complementarily with some regions of the guide nucleic acid on the nucleic acid. Can be.
  • uracil (U) and thymine (T) may be included.
  • uracil (U) and adenine (Adeinie; A) may be included.
  • the target site may be an endo-polynucleotide present on the genome.
  • the target site may be an exo-polynucleotide inserted into the genome.
  • the CRISPR / enzyme system is a protein that can interact with a target site on an animal genome or a target site of an exo-polynucleotide to cut a portion of the target site. It means a complex containing.
  • the CRISPR / enzyme system may comprise guide nucleic acid and RNA-guided endonuclease.
  • the term 'guide nucleic acid' refers to a nucleic acid capable of binding to a target site of an animal genome in a cell or a target site of an exo-polynucleotide.
  • the term 'guide nucleic acid' of the present application includes a guide nucleic acid composed of a single chain guide nucleic acid (single strand guide nucleic acid) and two or more strands of nucleic acid (nucleic acid).
  • Single chain guide nucleic acid may comprise a gRNA.
  • the gRNA is any one of a protospacer domain, a first complementary domain, a second complementary domain, a proximal domain, or a tail domain. It may contain the above.
  • the protospacer domain is a domain comprising a base sequence capable of complementary binding to a portion of a target site of an animal genome or a portion of a target site of an exo-polynucleotide.
  • the first complementary domain and the second complementary domain are domains capable of complementary binding to each other to interact with RNA-guided endonuclease.
  • the second complementary domain (second complementary domain) may be located downstream of the first complementary domain.
  • the proximal domain may be located downstream of the second complementary domain.
  • the tail domain may be located at the 3 'end of the gRNA.
  • Dual gRNAs may include crRNA and tracrRNA.
  • the crRNA may comprise a protospace domain and a first complementary domain.
  • the tracrRNA may comprise a second complementary domain (second complementary domain), a proximal domain, and a tail domain.
  • 'RNA-guided endonuclease' of the present application is a polypeptide or protein comprising a domain capable of interacting with a polynucleotide and a domain capable of cleaving an intermediate of the polynucleotide.
  • RNA-guided endonucleases may include, but are not limited to, SpCas9, CjCas9, StCas9, SaCas9, NmCas9, Cpf1 proteins, and mutants thereof.
  • RNA-guided endonucleases may include, but are not limited to, dead Cas9, Cas9 nickase, eSpCas9, and SpCas9-HF1 depending on the modified / engineered purpose.
  • RNA-guided endonucleases can interact with nucleic acids to cut double strands.
  • the RNA-guided endonuclease may interact with a nucleic acid to cut one of the double strands.
  • the RNA-guided endonuclease may interact with the nucleic acid but not cut the nucleic acid.
  • RNA-guided endonuclease When the RNA-guided endonuclease interacts with a nucleic acid to cut a double strand or cut a strand, a nucleotide or polynucleotide insertion may occur at the cut portion. Alternatively, when the RNA-guided endonuclease interacts with nucleic acid to cut double strands or cut one strand, nucleotide or polynucleotide deletion may occur at the cut region. have.
  • the target site of the animal genome in the cell or the target site of the exo-polynucleotide may be a base sequence adjacent to the 5 'end or 3' end of the protospacer-adjacent motif (PAM) sequence.
  • PAM protospacer-adjacent motif
  • the PAM sequence may include, but is not limited to, NGG, NNGRRT, NNAGAAW, NNNNGATT, NNNVRYAC, and TTN.
  • N may be any one of A, T, U, G, or C.
  • V may be any one of A, C, or G.
  • W may be either A or T.
  • Y may be either C or T.
  • the PAM sequence may vary depending on RNA-guide endonucleases.
  • SpCas9 or a mutant thereof may have a PAM sequence of NGG.
  • SaCas9 or a mutant thereof may have a PAM sequence of NNGRRT.
  • StCas9 or a mutant thereof may have a PAM sequence of NNAGAAW.
  • NmCas9 or a mutant thereof may have the PAM sequence NNNGATT.
  • CjCas9 or a mutant thereof may have a PAM sequence of NNNVRYAC.
  • Cpf1 and its mutants may have a PAM sequence of TTN.
  • the term 'transposon' in the present application is a polynucleotide capable of repositioning on the animal genome in a cell.
  • the term 'transposon' of the present application means a polynucleotide capable of repositioning between a nucleic acid present in a cell and an animal genome.
  • the term 'transposon' refers to a polynucleotide which can be translocated within an animal genome in a cell.
  • the term 'transposon' also refers to a polynucleotide which can be translocated between a nucleic acid and an animal genome in a cell.
  • the transposon can be divided into a Class I transposon (retrotransposon) and a Class II transposon (DNA transposon).
  • Class I transposons are those in which a RNA is transcribed from a nucleic acid or transposon DNA on an animal genome in a cell, and then the DNA transcribed from the RNA is transferred to another location on the animal genome. It works by being inserted.
  • Class II transposons cut the transposon DNA on a nucleic acid or an animal genome in a cell and insert the cut transposon DNA at another location on the animal genome. Works the way it works.
  • the Class II transposon may comprise a first polynucleotide at the 5 'end, a second polynucleotide at the 3' end, and a third polynucleotide.
  • the first polynucleotide and the second polynucleotide may include an inverted terminal repeat (ITR) sequence.
  • the third polynucleotide may be located between the first polynucleotide and the second polynucleotide.
  • the third polynucleotide may comprise an exo-polynucleotide.
  • the third polynucleotide may comprise a polynucleotide encoding a transposase.
  • the term 'transposon' is assumed to be a Class II transposon, although the term 'transposon' is interpreted as a Class I transposon. If there is no problem, it should not be construed as limited to Class II transposons.
  • the term 'transposase' of the present application is a class II transposon by interacting with an ITR sequence located at both ends of a class II transposon located on a nucleic acid or an animal genome in a cell.
  • Class II transposon or a class II transposon (Class II transposon) refers to a protein (protein) that can be inserted on the animal genome (animal genome).
  • the transposase may include hobo / Ac / Tam, P element, Sleeping Beauty (SB), Frog Prince, Hsmar1, Hsmar2, piggyBac (PB), Tol2, and mutants thereof. This is not restrictive.
  • the transposase may cleave a transposon present on a nucleic acid or an animal genome in a cell.
  • the transposase may insert a Class II transposon on an animal genome.
  • the position at which the transposase inserts a Class II transposon on the animal genome may be irrelevant to the base sequence on the animal genome.
  • the position at which the transposase inserts a Class II transposon on the animal genome is located on an animal genome recognizable by the transposase. It may be determined according to a specific base sequence. For example, in the case of Sleeping Beauty, a transposon may be inserted by recognizing a TA sequence on an animal genome. PiggyBac can also recognize a TTAA sequence on the animal genome and insert a transposon.
  • the transposase and the Class II transposon may be used for inserting exo-polynucleotides into an animal genome in a cell.
  • an intracellular animal genome is provided by delivering cells with exo-polynucleotides containing 5B and 3 'end of the ITR sequences that can interact with piggyBac transposase and piggyBac.
  • the exo-polynucleotide may be inserted into the exo-polynucleotide.
  • exo-polynucleotides that can be inserted into the animal genome in the cell. Therefore, by inserting a plurality of exo-polynucleotides encoding the target protein in the animal genome of the cell (animal genome) it can increase the expression amount of the target protein of the cell.
  • the position at which the exo-polynucleotide is inserted by transposase may be a position that does not interfere with gene expression of the cell.
  • a transposon can be inserted at the intron, 5 'UTR, or 3' UTR position of the animal genome.
  • the term 'site-specific recombination' of the present application refers to a pair of base sequences in which a pair of two base sequences identical or identical on a nucleic acid or animal genome is paired. Interchange occurs.
  • the base sequence in which the interchange occurs is called a recombinase recognition site (RRS).
  • RRS recombinase recognition site
  • SSR site-specific recombinase
  • the recombinase recognition site (RRS) of the present application may include, but is not limited to, loxp, rox, FRT, attP, attB, and mutants thereof.
  • the loxp mutant may include, but is not limited to, loxp66, loxp71, loxp72, loxp2722, loxp5171, and loxpm2.
  • the rox mutant may include, but is not limited to, rox4R, rox6R, and rox2N.
  • the FRT mutant may include, but is not limited to, F3, F5, F10, F11, F12, F13, F14, F15, and F16.
  • the RRS may be paired with two to interact with the SSR.
  • the loxp or loxp mutant may be a pair of two identical RRS.
  • the rox or rox mutant may be a pair of two identical RRS.
  • the FRT or FRT mutant may be a pair of two identical RRS.
  • the attP may be paired with attB.
  • Site-specific recombinase (SSR) or recombinase of the present application is Cre, Dre, Flp, KD, B2, B3, lambda, HK022, HP1, gamma delta, ParA, Tn3 , Hin, Gin, Pin, phiC31, Bxb1, R4, or a mutant thereof, but is not limited thereto.
  • the SSR may interact with a pair of RRSs. Cre or Cre mutants can specifically interact with loxp or loxp mutants. Dre or Dre mutants can specifically interact with rox or rox mutants. Flp or Flp mutants can specifically interact with FRT or FRT mutants. PhiC31 or phiC31 mutants can specifically interact with attP and attB.
  • Site-specific recombination of the present application may include insertion, removal, inversion, and replacement.
  • the site-specific recombination is reversible or irreversible.
  • the first nucleic acid When one of the pair of RRSs is located in the first nucleic acid (nucleic acid) and the other is located in the second nucleic acid (nucleic acid), the first nucleic acid through interaction with the SSR specific to the pair of RRS Insertion of the second nucleic acid into a nucleic acid may occur.
  • the second nucleic acid may be removed through interaction with the SSR.
  • inversion is reversible, inversion of the polynucleotides located between the pair of RRSs may occur again.
  • RRS A and RRS B are paired and RRS C and RRS D are paired
  • the RRS A and RRS C are located in the first nucleic acid
  • the RRS B and RRS D are the second nucleic acid ( nucleic acid).
  • RRS C may be located downstream from RRS A in the first nucleic acid
  • RRS D may be located downstream from RRS B in the second nucleic acid.
  • interaction with the SSR specific to the first pair of RRSs and interaction with the SSR paired with the second pair of RRSs may occur.
  • the replacement of polynucleotides located between RRS A and RRS C and polynucleotides located between RRS B and RRS D may occur.
  • replacement of the polynucleotide located between the RRS A and RRS C and the polynucleotide located between the RRS B and the RRS D may occur again.
  • the term 'marker gene' refers to a gene that is inserted into an animal genome in a cell in order to select a cell in which the intended transformation has been performed.
  • An exo-polynucleotide comprising a polynucleotide encoding a marker gene is introduced into a cell, and then the exo-polynucleotide is inserted into an animal genome ( When inserted, the marker gene may be expressed in cells to help select cells.
  • the marker gene is an antibiotic resistance gene, an antigen gene, a luciferase gene, a beta-galactosidase gene, a fluorescent protein gene.
  • fluorescent protein gene fluorescent protein gene
  • surface protein gene surface marker gene
  • suicide gene suicide gene
  • an antibiotic compound may be cultured together to perform cell selection.
  • the antibiotic compound may include, but is not limited to, ampicillin, chloramphenicol, tetratracycline, and kanamycin.
  • an antibody capable of specifically interacting with the antigen Antibodies can be cultured together to allow cell selection.
  • the antigen may include a surface antigen.
  • the surface antigen may include a CD molecule.
  • the antibody may interact with magnetic particles or fluorophores.
  • cell selection may be performed by culturing luciferin together.
  • the marker gene is a beta-galactosidase gene
  • it is cultured together with X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Cell selection can be performed.
  • fluorescent protein gene Genes encoding fluorescent proteins (fluorescent protein gene, gene encoding fluorescent protein)
  • the fluorescent protein may include a green fluorescent protein (hereinafter referred to as GFP), a yellow fluorescent protein (hereinafter referred to as YFP), and a red fluorescent protein (hereinafter referred to as RFP). This is not restrictive.
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • the marker gene is a suicide gene
  • cell selection may be performed by culturing a prodrug paired with the suicide gene.
  • the suicide gene is a thymidine kinase gene, cytosine deaminase gene, cytochrome P450 gene, nitroreductase gene, purine nucleo Side phosphorylase gene (purine nucleoside phosphorylase gene), and carboxypeptidase G2 gene (carboxypeptidase G2 gene) may include, but is not limited thereto.
  • the prodrugs are acyclovir, ganciclovir, 5-fluorocytosine, cyclophosphamide, ifosfamide, 5- [aziridine- 1-yl] -2,4-dinitrobenzamide (5- [aziridin-1-yl] -2,4-dinitrobenzamide) (CB1954), 6-methylpurine-2-deoxyriboside (6-methylpurine- 2-deoxyriboside) (MeP-dR), arabinofuranosyl-2-fluoroadenine monophosphate (F-araA), and N, N-[(2-chloroethyl) (2 -Mesyloxyethyl) aminobenzoic acid] (N, N-[(2-chloroethyl) (2-mesyloxyethyl) amino] benzoic acid) (CMDA), but is not limited thereto.
  • CB1954 6-methylpurine-2-deoxyriboside
  • MeP-dR 6-methylpur
  • the term 'expression control element' in the present application includes a transcriptional control element, a post-transcriptional processing control element, a translational control element, and a post-translational modification control element.
  • 'transcriptional regulatory element' of the present application means a substance or base sequence capable of initiating, promoting, inhibiting or terminating the synthesis of mRNA from a polynucleotide encoding RNA or a polypeptide.
  • the transcriptional regulatory element can be an enhancer, a silencer, a repressor, an activator, an inhibitor, a promoter, a transcription termination codon, and a loxp-transcription codon-loxp (hereinafter LTL). It may include, but is not limited thereto.
  • the transcription termination codon may include, but is not limited to, a poly T sequence (a term “poly T sequence” is used interchangeably with the term “poly A sequence”) and an AATAAA sequence.
  • post-transcriptional regulatory elements refers to chemical compounds, polynucleotides, or enzymes capable of modifying the structure of mRNA synthesized from polynucleotides.
  • the post-transfer process control element is a chemical that can cause 5 'capping, 3' cleavage, 3 'polyadenylation, or splicing ( chemical compounds), polynucleotides, or enzymes, but is not limited thereto.
  • 'translational regulatory element' of the present application means a substance or base sequence capable of initiating, promoting, inhibiting or terminating the synthesis of a polypeptide from an mRNA.
  • the translation control element may be a 3 'untranslated region (hereinafter referred to as 3' UTR), a 5 'untranslated region (hereinafter referred to as 5'UTR), exon, intron, initiation codon, termination codon, or coza Sequence, IRES, polynucleotides encoding 2A peptide (peptide), may include, but not limited to, loxp-stop codon-loxp (hereinafter LSL).
  • post-translational modification regulatory element refers to a chemical compound, polynucleotide, or enzyme that can cause modification to the structure of a polypeptide synthesized from mRNA. .
  • the post-translational modification regulatory elements are chemicals that can cause glycosylation, folding, ubiquitination, sumoylation, acetylation, or phosphorylation of polypeptides transcribed and translated from the polynucleotides. It may include, but is not limited to, a chemical compound or an enzyme.
  • a 'promoter' of the present application is a nucleic acid sequence that interacts with an RNA polymerase on a nucleic acid to initiate transcription.
  • the 'promoter' of the present application includes a constitutive promoter, a tissue-specific promoter, and an inducible promoter.
  • constitutive promoter refers to a promoter that initiates transcription regardless of environmental changes in the cell.
  • the constitutive expression promoter may include, but is not limited to, a CMV promoter, a CAG promoter, and a U6 promoter.
  • tissue-specific promoter of the present application means a promoter capable of initiating transcription only in a specific tissue of an animal.
  • the tissue-specific promoter may include, but is not limited to, a mammary tissue or a reproductive organ-specific promoter.
  • Promoters specific to the mammary gland are alpha-casein promoter, beta-casein promoter, kappa-casein promoter, and mu-casein promoter. , And beta-lactoglobulin promoters, but are not limited thereto.
  • Reproductive organ specific promoters may include, but are not limited to, ovarian-specific promoters and testis-specific promoters.
  • inducible promoter refers to a promoter that initiates transcription in response to environmental or external environmental changes in a cell.
  • the inducible promoter may include, but is not limited to, a chemically inducible promoter, a temperature inducible promoter, and a light inducible promoter.
  • the chemically inducible promoter may be an antibiotic-inducible promoter, an alcohol-inducible promoter, a steroid-inducible promoter, and a metal-induced promoter. -inducible promoter), but is not limited thereto.
  • the antibiotic-inducible promoter may include, but is not limited to, a Tet-on promoter and a Tet-off promoter.
  • the steroid-inducible promoter includes, but is not limited to, an estrogen-inducible promoter.
  • the metal-inducible promoter may include a copper-inducible promoter, but is not limited thereto.
  • the temperature inducible promoter may include, but is not limited to, a heat shock-inducible promoter and a cold shock-inducible promoter.
  • the high temperature shock-promoter may include, but is not limited to, an Hsp promoter.
  • the term 'delivery' of the present application may mean introducing exo-polynucleotides into organs, tissues, cells, or intracellular organelles of living organisms.
  • the term 'delivery' of the present application may mean introducing a polypeptide or protein into an organ, tissue, cell, or intracellular organelle of a living organism.
  • the term 'delivery' may be used interchangeably with the term 'providing'.
  • the delivery may include non-viral delivery.
  • the non-viral delivery may use a naked nucleic acid vector.
  • the naked nucleic acid vector may include a circular nucleic acid vector and a linear nucleic acid vector.
  • the circular nucleic acid vector may include, but is not limited to, a plasmid vector.
  • the non-viral delivery may use a non-viral vector.
  • the non-viral vector may include artificial chromosomes, liposomes, polymers, lipid-polymer hybrids, inorganic nanoparticles, And organic nanoparticles, but are not limited thereto.
  • the non-viral delivery may include microinjection, gene gun, electroporation, sonoporation, photoporation and magnetofection. magnetofection), and hydroporation, but is not limited thereto.
  • the gene delivery may include viral delivery.
  • the viral delivery may use an RNA-based viral vector.
  • the RNA-based viral vector may include an oncoretroviral vector, a lentiviral vector, and a human foamy viral vector. It is not limited.
  • the viral delivery may use a DNA-based viral vector.
  • the DNA-based viral vector may be an adenoviral vector, an adeno-associated viral vector, or an Epstein-Barr viral vector. ), Herpes simplex viral vector, and poxviral vector, but are not limited thereto.
  • the term 'microinjection' refers to the injection of a substance into organs, tissues, cells, or intracellular organelles of a living organism.
  • the material may include a chemical compound, a polynucleotide, or a polypeptide.
  • microinjection' may include gamete microinjection, zygote microinjection, embryo microinjection, and somatic cell microinjection. have.
  • the term 'gamete microinjection' of the present application means microinjection of a material including polynucleotides into a gamete.
  • the term 'gamete microinjection' of the present application refers to a technique for obtaining a transgenic animal through microinjection of a material containing polynucleotides into a gamete and then through a fertilization step and a differentiation step. Include.
  • the term 'zygote microinjection' of the present application refers to microinjection of a material including polynucleotides in a zygote.
  • the term 'zygote microinjection' of the present application includes a technique of obtaining a transgenic animal through microinjection of a substance containing polynucleotide in a zygote and then through a differentiation step.
  • the term 'embryo microinjection' of the present application refers to microinjection of a material including polynucleotides into an embryo.
  • the term 'embryo microinjection' of the present application includes a technique of obtaining a transgenic animal through microinjection of a material containing polynucleotides into an embryo and then through a differentiation step.
  • Somatic cell microinjection' of the present application means microinjection of a material including polynucleotides into a somatic cell.
  • 'nuclear transfer means removing a nucleus from an oocyte and then introducing a donor nucleus obtained from another cell into the oocyte.
  • SCNT 'somatic cell nuclear transfer
  • the term 'nuclear transfer' refers to removing a nucleus from an animal's oocyte, introducing a donor nucleus from an animal cell, reprogramming, And a technique for obtaining an animal comprising genetic information identical to that of an animal cell including a donor nucleus through a differentiation step and a surrogate mother transplantation step.
  • the term 'somatic cell nuclear transfer (SCNT)' of the present application refers to the somatic cell through the aforementioned steps when the animal cell including the donor nucleus is a somatic cell. Techniques for obtaining an animal containing the same genetic information as
  • transgenic animal can be produced by delivery to a cell including the donor nucleus.
  • transgenic animals may be prepared through somatic cell microinjection and then through somatic cell nuclear transfer (SCNT).
  • SCNT somatic cell nuclear transfer
  • Animals of the present application may include non-human animals.
  • the animal may comprise a mammal.
  • the mammal may comprise an ungulate.
  • the ungulate may include a perissodactyl.
  • the base may include horses, but is not limited thereto.
  • the ungulate may include artiodactyl.
  • the cow products may include, but are not limited to, pigs, deer, cattle, sheep and goats.
  • the mammal may comprise a rodent.
  • the rodent may include a mouse and a rat, but is not limited thereto.
  • the mammal may comprise a lagomorph.
  • the rabbit neck may include rabbit and hare, but is not limited thereto.
  • 'bioreactor' of the present application refers to an organism capable of causing a biological reaction or a chemical reaction occurring in the organism.
  • the organism can include cells, cell lines, and animals. Such organisms may include transformed cells, transformed cell lines, and transformed animals.
  • the bioreactor may be a transgenic rat.
  • the bioreactor may be a transgenic rat.
  • the bioreactor may be a transgenic cow.
  • the bioreactor can be used to produce the desired material.
  • the target substance may include a target protein.
  • the bioreactor is a transgenic cow knocked on the human albumin gene, the transgenic cow can be used to produce human albumin.
  • Toolbox ' or ' toolbox ' in the present application means an exo-polynucleotide which can be inserted into or can be inserted into an animal genome in a cell.
  • the cell into which the toolbox can be inserted may be a transformed cell. Or may not be a transformed cell.
  • the term 'toolbox' of the present application may mean an exo-polynucleotide for transformation of an animal genome in a cell.
  • the toolbox may include a polynucleotide encoding a protein of interest in an animal genome in a cell.
  • the Toolbox encodes a component of an artificial nuclease or a component of an artificial nuclease capable of site-specific transformation of an animal genome in a cell.
  • Polynucleotides may be included.
  • the toolbox may include a transposon.
  • the toolbox may include a first end region at the 5 'end, a second end region at the 3' end, and a core region located between the first end region and the second end region.
  • the first end region and the second end region may comprise at least one editing enabling component.
  • the first terminal region and the second terminal region may comprise the same polynucleotide.
  • the first terminal region and the second terminal region may include different polynucleotides.
  • the first terminal region and the second terminal region may include an ITR sequence, and the core region may include a polynucleotide existing between the ITR sequences.
  • the core region may include an editing enabling component.
  • the core region may comprise a polynucleotide encoding a protein or RNA.
  • the core region may comprise a polynucleotide encoding a non-functional peptide.
  • the core region may comprise a polynucleotide encoding an artificial intron.
  • the core region may comprise a polynucleotide encoding a non-functional RNA.
  • the core region may comprise polynucleotides that are not transcribed.
  • the core region may comprise polynucleotides that are not translated.
  • the core region may comprise expression control elements.
  • the core region may include a promoter.
  • the toolbox may include one or more polynucleotides that encode the RRS.
  • the RRS may include, but is not limited to, loxp, rox, FRT, attP, attB, and mutations thereof.
  • the loxp mutations may include, but are not limited to, loxp66, loxp71, loxp72, loxp2722, loxp5171, and loxpm2.
  • the rox mutations may include, but are not limited to, rox4R, rox6R, and rox2N.
  • the FRT mutations can include, but are not limited to, F3, F5, F10, F11, F12, F13, F14, F15, and F16.
  • the toolbox may comprise one or more ITR sequences.
  • the ITR sequence may interact with hobo / Ac / Tam, P element, Sleeping Beauty (SB), Frog Prince, Hsmar1, Hsmar2, piggyBac (PB), Tol2, or mutations thereof.
  • the toolbox may comprise one or more artificial nuclease target sites.
  • the artificial nuclease target site may comprise a target site of the CRISPR / enzyme system.
  • the target site may be a nucleotide sequence adjacent to the 5 'end or 3' end of the PAM sequence.
  • the toolbox may comprise a portion of an artificial nuclease target site in one terminal region.
  • the toolbox may comprise a PAM sequence and another portion of the artificial nuclease target site in the other terminal region.
  • the toolbox may comprise one or more polynucleotides encoding a protein.
  • the kind of the target protein is not limited as long as it can be produced in a cell or an animal subject to the transformation technique disclosed in the present application.
  • the protein of interest may comprise one component of an artificial nuclease.
  • an artificial nuclease for example, zinc finger nucleases (ZFNs), talens (TALENs), RNA-guided endonucleases, or modified / engineered forms thereof. May be, but is not limited thereto.
  • the target protein may include a non-human animal derived protein.
  • a non-human animal derived protein for example, non-human albumin, non-human interleukin, non-human insulin, non-human erythropoietin, Non-human antibodies, non-human omega3s, or modified / engineered forms thereof, but are not limited thereto.
  • the target protein may include a human-derived protein.
  • human albumin human interleukin, human insulin, human erythropoietin, human gamma chain, human delta chain
  • Human alpha chain human mu chain
  • human epsilon chain human kappa chain
  • human lambda chain or their It may include, but is not limited to, a modified / engineered form.
  • the toolbox may include one or more polynucleotides that encode a marker gene.
  • the marker gene is an antibiotic resistance gene, an antigen gene, a luciferase gene, a beta-galactosidase gene, a fluorescent protein gene, and a suicide gene. (suicide gene) may be included, but is not limited thereto.
  • the toolbox may include one or more polynucleotides that encode the SSR.
  • the SSR may include Cre, Dre, Flp, KD, B2, B3, lambda, HK022, HP1, gamma delta, ParA, Tn3, Hin, Gin, Pin, phiC31, Bxb1, R4, or mutations thereof, This is not restrictive.
  • the toolbox may comprise one or more polynucleotides encoding a transposase.
  • the transposase may include, but is not limited to, hobo / Ac / Tam, P element, Sleeping Beauty (SB), Frog Prince, Hsmar1, Hsmar2, piggyBac (PB), Tol2, and mutations thereof.
  • the toolbox may comprise one or more polynucleotides encoding endonucleases.
  • the endonuclease may include, but is not limited to, zinc finger nuclease (ZFN), talene (TALEN), and RNA-guided endonucleases.
  • the toolbox may comprise one or more polynucleotides that encode RNA-guide endonucleases.
  • the RNA-guided endonuclease may comprise a Cas9 or Cas9 mutation.
  • the RNA-guide endonuclease may include, but is not limited to, Cpf1 or Cpf1 mutations.
  • the toolbox may comprise one or more polynucleotides encoding any one or more of crRNA, tracrRNA, and gRNA.
  • the toolbox may comprise one or more polynucleotides encoding a non-functional polypeptide.
  • the polynucleotide encoding the non-functional polypeptide may be a part of a polynucleotide encoding a protein of interest, a part of a marker gene, a part of a polynucleotide encoding a position-specific recombinase, or a trans Part of a polynucleotide encoding a posase, and part of a polynucleotide encoding an endonuclease, but is not limited thereto.
  • Polynucleotides encoding the non-functional polypeptide may include a stop codon.
  • the polynucleotides encoding the non-functional polypeptide may comprise LSL.
  • the polynucleotide encoding the non-functional polypeptide may comprise a polynucleotide encoding a 2A peptide.
  • the polynucleotides encoding the non-functional polypeptide may comprise an IRES.
  • the toolbox may comprise one or more polynucleotides that encode untranslated RNA.
  • the polynucleotides encoding the untranslated RNA may comprise a guide nucleic acid.
  • the polynucleotides encoding the untranslated RNA may comprise an AATAAA sequence.
  • the polynucleotides encoding the untranslated RNA may comprise poly T.
  • the polynucleotides encoding the untranslated RNA may not comprise an initiation codon.
  • the polynucleotides encoding the untranslated RNA may not comprise a Kozak sequence.
  • the toolbox may comprise one or more polynucleotides that are not transcribed.
  • the non-transcribed polynucleotide may be a promoter.
  • the base sequence of the non-transcribed polynucleotide may be a base sequence that is not present on the animal genome in the cell.
  • position-specific transformation can be performed without interacting with the animal genome in the cell.
  • the base sequence of the non-transcribed polynucleotide may be identical to some base sequence of the polynucleotide encoding a protein or RNA that the cell can normally express.
  • the promoter may not be located upstream of the non-transcribed polynucleotide.
  • the non-transcribed polynucleotide can be utilized as an artificial nuclease target site.
  • gRNAs and Cas9s that can bind the same or complementary to a portion of the nontranscribed polynucleotides are introduced into the cell and the Site-specific transformation can be caused on non-transcribed polynucleotides.
  • the toolbox may include one or more artificial introns.
  • the artificial intron may be included in the toolbox together with or alone with a polynucleotide encoding a protein of interest.
  • the artificial intron can be located within the transcription unit (unit) of the polynucleotide encoding the protein of interest.
  • the artificial intron may include a splice donor site at the 5 'end and a splice acceptor site at the 3' end.
  • One or more polynucleotides encoding an RRS may be included between a splice donor site and a splice acceptor site of the artificial intron.
  • the artificial intron may comprise a stop codon.
  • the artificial intron may include an enhancer.
  • the artificial intron can be removed by splicing.
  • the artificial intron is a) derived from a natural intron of the gene of interest itself or b) modified by substitution, deletion and / or insertion of nucleotides, c) a natural intron from different genes of interest, d) a different intron E) chimeric introns consisting of different intron sequences derived from one or more natural intron sequences of the target gene and / or different genes, f) a novel synthesized synthetase intron and g) any combination of the above. Can be.
  • the artificial intron can increase or decrease the expression of a polynucleotide encoding a protein of interest.
  • the artificial intron may increase or decrease the expression of polynucleotides in the genome.
  • the toolbox may comprise one or more expression control elements.
  • the expression control element may comprise a transcriptional control element.
  • the transcriptional regulatory element is as set forth above.
  • the expression control element may comprise a post-transcriptional processing control element.
  • the post-transfer processing control element is as previously presented.
  • the expression control element may comprise a translational control element.
  • the translation control element is as described above.
  • the expression control element may comprise a post-translational modification regulatory element.
  • the post-translational modification regulatory elements are as set forth above.
  • the toolbox may include a promoter.
  • the promoter may comprise a constitutive expression promoter.
  • the constitutive expression promoter is as shown above.
  • the promoter may comprise a tissue specific promoter.
  • the tissue specific promoter is as shown above.
  • the promoter may comprise an inducible promoter.
  • the induction promoter is as shown above.
  • the first terminal region and the second terminal region of the toolbox may comprise an ITR sequence.
  • the core region of the toolbox may include at least one recombinase recognition site.
  • the toolbox can provide a location for insertion or exchange of exo-polynucleotides without adversely affecting gene expression of the animal genome in the cell.
  • the first terminal region and the second terminal region of the toolbox may comprise an ITR sequence.
  • the core region of the toolbox may comprise at least one artificial nuclease target site.
  • the toolbox can provide a location for insertion or exchange of exo-polynucleotides without adversely affecting gene expression of the animal genome in the cell.
  • the first terminal region and the second terminal region of the toolbox may comprise an ITR sequence.
  • the core region of the toolbox may comprise a polynucleotide encoding an RNA-guide endonuclease.
  • the core region of the toolbox may include at least one recombinase recognition site.
  • a polynucleotide encoding a guide nucleic acid may be inserted into the toolbox using the at least one recombinase recognition site. Using the recombinase recognition site, the target site at which site-specific transformation will occur can be altered.
  • the at least one recombinase recognition site may comprise recombinase recognition site 1 (RRS 1) and recombinase recognition site 2 (RRS 2).
  • the recombinase recognition site 1 (RRS 1) is located upstream of the polynucleotide encoding the RNA-guide endonuclease
  • the recombinase recognition site 2 (RRS 2) is the RNA-guide endonuclease. May be located downstream of the polynucleotide encoding.
  • the recombinase recognition site 1 (RRS 1) and recombinase recognition site 2 (RRS 2) can be used to exchange polynucleotides encoding RNA-guide endonucleases in the toolbox with other exo-polynucleotides.
  • Polynucleotide-recombinase recognition site-expressing regulatory element-ITR sequence encoding a polynucleotide-guided nucleic acid encoding an ITR sequence-RNA-guide endonuclease
  • the first terminal region and the second terminal region of the toolbox may comprise an ITR sequence.
  • the core region of the toolbox may comprise a polynucleotide encoding an RNA-guide endonuclease.
  • the core region of the toolbox may comprise a polynucleotide encoding a guide nucleic acid.
  • the core region of the toolbox may include at least one recombinase recognition site.
  • the core region of the toolbox may include at least one or more of an element regulating the expression of RNA-guide endonucleases or an element regulating the expression of guide nucleic acid.
  • the at least one recombinase recognition site may be located upstream or downstream of the polynucleotide encoding the RNA-guide endonuclease.
  • the at least one recombinase recognition site can be used to insert or remove elements that regulate the expression of a polynucleotide encoding an RNA-guided endonuclease.
  • the at least one recombinase recognition site may be located upstream or downstream of the polynucleotide encoding the guide nucleic acid.
  • the at least one recombinase recognition site may be used to insert or remove elements that control the expression of a polynucleotide encoding a guide nucleic acid.
  • the at least one recombinase recognition site may comprise recombinase recognition site 1 (RRS 1) and recombinase recognition site 2 (RRS 2).
  • the recombinase recognition site 1 (RRS 1) is located upstream of the polynucleotide encoding the guide nucleic acid
  • the recombinase recognition site 2 (RRS 2) is downstream of the polynucleotide encoding the guide nucleic acid. It can be located at The recombinase recognition site 1 (RRS 1) and recombinase recognition site 2 (RRS 2) can be used to remove polynucleotides encoding the guide nucleic acid in the toolbox.
  • the first terminal region and the second terminal region of the toolbox may comprise an ITR sequence.
  • the core region of the toolbox may comprise a polynucleotide encoding at least one marker gene.
  • the at least one marker gene may comprise a suicide gene.
  • Marker genes may be knocked out through position-specific transformation into polynucleotides encoding the marker genes.
  • the first terminal region of the toolbox may comprise recombinase recognition site 1 (RRS 1).
  • the second terminal region of the toolbox may comprise recombinase recognition site 2 (RRS 2) paired with the recombinase recognition site 1 (RRS 1).
  • the core region of the toolbox may comprise a polynucleotide encoding a transposase.
  • the transposase may comprise an exclusion-only transposase.
  • the toolbox can be used to remove transposons contained in the animal genome in cells.
  • the first terminal region and the second terminal region of the toolbox may comprise an artificial nuclease target site.
  • the core region of the toolbox may comprise a first ITR sequence, a second ITR sequence, and an exo-polynucleotide.
  • the polynucleotide encoding the target protein may be located between the first ITR sequence and the second ITR sequence.
  • the toolbox can be used to insert exo-polynucleotides with site-specific transformation on the animal genome in cells. And only exo-polynucleotides can be removed with transposase while maintaining site-specific transformation on the animal genome in the cell.
  • the toolbox can be used to express proteins or nucleic acids necessary for the transformation of the animal genome in cells.
  • the toolbox may comprise a polynucleotide encoding Cas9.
  • the complex forms with Cas9 expressed in the cell to cause site-specific transformation at the target site on the animal genome Can be.
  • the toolbox can be used to knock genes on the animal genome in a cell.
  • the toolbox may include polynucleotides encoding human interleukin.
  • the toolbox can be inserted onto the bovine genome in the cell to allow the cell to express human interleukin.
  • the toolbox can be used to knock out genes present on the animal genome in a cell.
  • the toolbox can be inserted in the middle of a polynucleotide encoding the exon of bovine albumin on the bovine genome in the cell, preventing the cell from expressing bovine albumin in bovine. have.
  • the toolbox can provide a transformable site on the animal genome in a cell.
  • the toolbox may comprise a base sequence that may be the target site of an artificial nuclease.
  • the toolbox may include polynucleotides encoding green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • gRNA and Cas9 which targets some nucleotide sequences of the polynucleotides encoding green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • the toolbox can be delivered for insertion into the animal genome in the cell.
  • the nucleic acid delivery vector including the toolbox may be a naked nucleic acid vector, a non-viral vector, or a viral vector.
  • a first homology sequence may be located at the 5 'end of the first end region of the toolbox.
  • a second homology sequence may be located at the 3 ′ end of the second end region of the toolbox.
  • the first homology sequence (homology arm) may have the same base sequence as part of the animal genome in the cell.
  • the second homology arm may have the same nucleotide sequence as a part of the animal genome in the cell.
  • the first homology arm and the second homology arm may interact with a part of a genome in a cell. This interaction allows the toolbox to be inserted into the genome.
  • the first terminal region of the toolbox may comprise RRS 1.
  • the second terminal region of the toolbox may comprise RRS 2.
  • At least one recombinase may be provided for introducing the toolbox into a cell.
  • the recombinase may comprise a first recombinase capable of interacting with RRS 1.
  • the recombinase may comprise a second recombinase capable of interacting with RRS 2.
  • the first recombinase and the second recombinase may be identical to each other.
  • the first recombinase and the second recombinase may be Cre.
  • viral vector, or recombinase polypeptide can be used.
  • a nucleic acid including the toolbox and the at least one recombinase may be provided in separate forms.
  • a nucleic acid containing a toolbox can be delivered to a DNA plasmid vector and the recombinase can be delivered to a recombinase polypeptide.
  • a nucleic acid (nucleic acid) including the toolbox and the at least one recombinase may be provided as one delivery vector.
  • a polynucleotide encoding a recombinase and a nucleic acid containing a toolbox can be provided as a single DNA plasmid vector.
  • the first terminal region and the second terminal region of the toolbox may comprise an ITR sequence.
  • Transposase may be provided for introducing the toolbox into a cell.
  • the transposase may interact with the ITR sequence.
  • a naked nucleic acid vector, a non-viral vector, a viral vector comprising a polynucleotide encoding the transposase to introduce the transposase into a cell vector), or transposase polypeptide may be used.
  • Nucleic acid (nucleic acid) including the toolbox and the transposase can be introduced into the cell in a separate form.
  • the nucleic acid containing the toolbox (nucleic acid) can be delivered to the DAN plasmid vector and the transposase can be delivered to the polypeptide.
  • Nucleic acid (nucleic acid) containing the toolbox and the transposase can be introduced into the cell as a separate vector.
  • a nucleic acid containing a toolbox may be delivered as a DNA plasmid vector
  • the transposase may be delivered as a separate DNA plasmid vector comprising a polynucleotide encoding a transposase.
  • Nucleic acid (nucleic acid) including the toolbox and the transposase can be introduced into the cell as a vector.
  • a nucleic acid containing a toolbox (nucleic acid) and a polynucleotide encoding a transposase can be delivered in one DNA plasmid vector.
  • a first homology sequence may be located at the 5 'end of the first end region of the toolbox.
  • a second homology arm may be located at the 3 'end of the second end region of the toolbox.
  • the first homology sequence (homology arm) may have the same base sequence as part of the animal genome in the cell.
  • the second homology arm may have the same nucleotide sequence as a part of the animal genome in the cell.
  • Any one of a nucleic acid delivery vector containing the toolbox may be used to introduce the toolbox into a cell.
  • Artificial nucleases can be provided for introducing the toolbox into a cell.
  • the artificial nuclease may act specifically on an artificial nuclease target site present on the animal genome.
  • An artificial nuclease target site present on the animal genome is located on the same or complementary nucleotide sequence as the first homology arm, or the same or complementary to the second homology arm. Or a base sequence identical or complementary to the first homology arm and a base sequence identical or complementary to the second homology arm.
  • the at least one artificial nuclease comprises a naked nucleic acid vector, a non-viral vector, a viral vector comprising a polynucleotide encoding each component of the artificial nuclease. (viral vector), an artificial nuclease protein, a complex comprising an artificial nuclease protein, or a combination thereof.
  • a complex comprising Cas9 protein and gRNA can be provided.
  • a naked nucleic acid vector may be provided comprising a polynucleotide encoding Cas9 and a polynucleotide encoding gRNA.
  • a nucleic acid including the toolbox and the at least one artificial nuclease may be provided in separate forms.
  • a nucleic acid containing a toolbox may be provided as a DNA plasmid vector
  • an artificial nuclease may be provided as a complex comprising a Cas9 protein and a gRNA.
  • the nucleic acid comprising the toolbox is provided as a DNA plasmid vector
  • the artificial nuclease is provided as a naked nucleic acid vector comprising a polynucleotide encoding Cas9 and a polynucleotide encoding a gRNA.
  • the nucleic acid including the toolbox and the artificial nuclease may be provided as one vector.
  • a nucleic acid comprising a toolbox, a polynucleotide encoding Cas9, and a polynucleotide comprising a gRNA can be provided in one DNA plasmid vector.
  • the first terminal region of the toolbox may comprise a first artificial nuclease target site.
  • the second terminal region of the toolbox may comprise a second artificial nuclease target site.
  • the first artificial nuclease target site and the second artificial nuclease target site may include the same base sequence.
  • At least one artificial nuclease may be provided for introducing the toolbox into a cell.
  • the artificial nuclease may comprise a first artificial nuclease that can specifically act on the first artificial nuclease target site.
  • the artificial nuclease may comprise a second artificial nuclease that can specifically act on the second artificial nuclease target site.
  • the first artificial nuclease and the second artificial nuclease may be identical to each other.
  • the at least one artificial nuclease includes a naked nucleic acid vector, a non-viral vector, a viral vector, an artificial nuclease protein, and an artificial nuclease protein, including a polynucleotide encoding each component of the artificial nuclease. It may be provided in any one of a complex, or a combination thereof.
  • the nucleic acid comprising the toolbox and the at least one artificial nuclease may be provided in separate forms to introduce the toolbox into a cell.
  • a nucleic acid comprising a toolbox may be provided as a DNA plasmid vector
  • an artificial nuclease may be provided as a complex comprising a Cas9 protein and a gRNA.
  • the nucleic acid comprising the toolbox is provided as a DNA plasmid vector
  • the artificial nuclease is provided as a naked nucleic acid vector comprising a polynucleotide encoding Cas9 and a polynucleotide encoding a gRNA. Can be.
  • the nucleic acid containing the toolbox and the at least one artificial nuclease may be provided as a vector to introduce the toolbox into a cell.
  • a nucleic acid comprising a toolbox, a polynucleotide encoding Cas9, and a polynucleotide comprising a gRNA can be provided in one DNA plasmid vector.
  • the animal genome in a cell may comprise one toolbox.
  • the one toolbox may be included in any one of the autosomal bodies of the animal genome.
  • the toolbox may be included in any one of the sex chromosomes of the animal genome.
  • the toolbox may be included in the X chromosome of the animal genome.
  • the toolbox may be included in the Y chromosome of the animal genome.
  • the animal genome in a cell may comprise two or more toolboxes.
  • the two or more toolboxes may be identical to each other.
  • One of the two or more toolboxes may be different from one of the others.
  • Different toolboxes may mean different toolbox sequences.
  • a first toolbox containing a polynucleotide encoding an RNA-guide endonuclease and a second toolbox containing a polynucleotide encoding a guide nucleic acid capable of binding to a target site may be viewed as different toolboxes. have.
  • the two or more polynucleotides may all be included in one chromosome.
  • the chromosome may be an autosomal or sex chromosome.
  • the two or more toolboxes may include at least a first toolbox and a second toolbox.
  • the first toolbox may be located on a first chromosome
  • the second toolbox may be located on a second chromosome different from the first chromosome.
  • the first chromosome may be an autosomal chromosome.
  • the first chromosome may be a sex chromosome.
  • the second chromosome may be an autosomal chromosome.
  • the second chromosome may be a sex chromosome.
  • the toolbox can be located between base sequences that can interact with the transposase.
  • Base sequences capable of interacting with the transposase may include TA and TTAA, but are not limited thereto.
  • the toolbox may be located on a polynucleotide that may be involved in protein or RNA expression on the animal genome in a cell.
  • the toolbox may be located in any of polynucleotides encoding a protein or RNA, a promoter of a polynucleotide encoding a protein or RNA, 5 ′ UTR, intron, exon, and 3 ′ UTR.
  • the toolbox may also knock out proteins or RNA on the animal genome in cells. For example, if the toolbox is located in the exon of the beta-lactoglobulin gene of the bovine genome, the cell containing the bovine genome is beta-lactoglobulin. May interfere with expression).
  • the toolbox may be located on a polynucleotide that is not involved in expressing a protein or RNA on the animal genome in a cell.
  • the toolbox may also be located in a safe harbor on the animal genome in the cell.
  • the safe harbor of the mouse genome may comprise the rosa26 locus, which is well known.
  • the safe harbor of the bovine genome may comprise a location on a bovine genome that is already well known.
  • the safe harbor of the bovine genome may include, but is not limited to, the locus of Table 1 below.
  • the locus may be located on the bovine chromosome between the gene closest to the 5 'end (5' gene) and the gene closest to the 3 'end (3' gene) shown in Table 1 below. have.
  • the toolbox may be capable of further transformation. It can be used as an artificial safe harbor.
  • a toolbox located in the safe harbor of a bovine genome contains loxp, it delivers exo-polynucleotides and Cre recombinase containing the loxp to affect protein or RNA expression in the genome in the cell. Exo-polynucleotides can be inserted into the toolbox without affecting.
  • the transformed cell comprising at least one toolbox may be a diploid cell.
  • the diploid cells include stem cells, somatic cells, ovarian stem cells, oogonium, primary oocytes, garden stem cells, spermatogonium, and first hair cells. (primary spermatocyte) and fertilized egg (zygote).
  • the transformed cell comprising at least one toolbox may be a haploid cell.
  • the haploid cell may comprise a second oocyte, an ovum, a second spermatocyte, and a sperm.
  • the transformed cell may comprise at least a pair of homologous chromosomes.
  • the at least one pair of homologous chromosomes may comprise a first chromosome and a second chromosome in a homologous chromosome relationship.
  • Transgenic cells comprising two or more toolboxes may be homozygote.
  • the type, number, and location of the toolboxes included in the first and second chromosomes may be the same.
  • the type and number of toolboxes included in the first and second chromosomes may be the same.
  • the first and second chromosomes may be of the same type of toolbox.
  • both the first and second chromosomes may not comprise a toolbox.
  • the transformed cell comprising two or more toolboxes may be heterozygote.
  • the first chromosome may not comprise a toolbox and the second chromosome may comprise at least one toolbox.
  • the second chromosome may not include the same toolbox as the toolbox included in the first chromosome.
  • Transgenic cells comprising at least one toolbox may form cell colonies.
  • the cell colony may be a cell population cultured from a single cell.
  • Each cell contained in a homologous cell colony may comprise one toolbox.
  • Each toolbox of each cell may be identical.
  • Each of the cells may be located in the same toolbox at the same location.
  • Each cell that a homologous cell colony contains may contain two or more toolboxes.
  • the type and location of the nth toolbox may be the same in each cell.
  • the two or more toolboxes may be identical to each other. Alternatively, one of the two or more toolboxes may be different from one of the remaining toolboxes.
  • the number, types, and types of toolboxes included in the first cell it means that the position is completely consistent with the number, type and location of the toolbox contained in the second cell.
  • Chimeric cell colonies refer to cell colonies other than homologous cell colonies.
  • Chimeric cell colonies may include cells having a genome that does not include a toolbox and cells that have a genome that includes at least one toolbox.
  • Chimeric cell colonies may comprise first and second cells having a genome containing at least one toolbox.
  • at least one or more of the toolbox included in the genome of the first cell and the type of toolbox included in the genome of the second cell, the number of toolboxes, and the locus of the toolbox are not the same.
  • the genome contained in the genome of the first cell i) the genome contained in the genome of the first cell.
  • the number of first toolboxes and the number of first toolboxes included in the genome of the second cell are different, or ii) the locus of the first toolbox included in the genome of the first cell and the genome of the second cell.
  • the cell colonies may be chimeric cell colonies when the locus of the first toolbox included is different.
  • a cell colony that contains a first toolbox in the genome of a first cell and another kind of second toolbox in the genome of a second cell
  • i) is included in the genome of the first cell.
  • the number of the first toolbox and the number of the second toolboxes included in the genome of the second cell are the same, ii) the locus of the first toolbox and the genome of the second cell included in the genome of the first cell.
  • the cell colonies may be chimeric cell colonies even if the positions of the second toolbox included in the same are all the same.
  • the first cell included in the genome of the first cell in which the first toolbox is included in the genome of the first cell and another kind of second toolbox is included in the genome of the second cell, i) the first cell included in the genome of the first cell.
  • the number of toolboxes is different from the number of second toolboxes included in the genome of the second cell, or ii) the locus of the first toolbox included in the genome of the first cell and the genome of the second cell are included.
  • the cell colonies may be chimeric cell colonies.
  • the term 'left' of the present application may be specified by any one or more of an endogenous gene closest to the 5 'end and an endogenous gene closest to the 3' end relative to the toolbox.
  • the term 'left of the toolbox' of the present application may be specified by any one or more of an endogenous gene closest to the 5 'end and an endogenous gene closest to the 3' end relative to the toolbox. have.
  • the positions of the first toolbox and the second toolbox are different. Further, when the endogenous gene closest to the 3 'end of the first toolbox and the endogenous gene closest to the 3' end of the second toolbox are different, the positions of the first toolbox and the second toolbox are different.
  • the core region of the toolbox may comprise an antibiotic resistance gene.
  • Animal cells comprising the toolbox can survive treatment with antibiotics. Therefore, the animal cell including the toolbox may be separated from the animal cell (animal cell) not containing the toolbox.
  • the core region of the toolbox may comprise a polynucleotide encoding an antigen or a nucleotide that can act as an antigen.
  • Animal cells comprising the toolbox may interact with antibodies specific for the antigen. Thus, animal cells comprising the toolbox can be distinguished from animal cells not containing the toolbox.
  • the core region of the toolbox may comprise polynucleotides encoding fluorescent proteins.
  • Animal cells including the toolbox may measure a fluorescence signal.
  • animal cells comprising the toolbox can be distinguished from animal cells not containing the toolbox.
  • the core region of the toolbox may comprise polynucleotides that encode a surface marker.
  • Animal cells comprising the toolbox may interact with antibodies specific for the surface markers.
  • the antibody may interact with magnetic particles or fluorophores. Therefore, animal cells including the toolbox may be distinguished from animal cells not containing the toolbox through magnetic properties or fluorescence signals.
  • the transgenic animal may comprise one or more transgenic cells comprising at least one toolbox.
  • Each cell included in a homologous transgenic animal may comprise one toolbox each.
  • Each toolbox of each cell may be identical.
  • Each of the cells may be located in one toolbox on the same chromosome.
  • Each cell contained by a homologous transgenic animal may comprise two or more toolboxes.
  • the chromosomes in which the n th toolbox is located may be identical in each cell.
  • the two or more toolboxes may be identical to each other. Alternatively, one of the two or more toolboxes may be different from one of the remaining toolboxes.
  • the homologous transgenic animal may comprise transgenic cells that are homozygous.
  • the homologous transgenic animal may comprise transgenic cells that are heterozygote.
  • Chimeric transgenic animals refer to transgenic animals other than homologous transgenic animals.
  • the chimeric transgenic animal may comprise homozygous transgenic cells.
  • the chimeric transgenic animal may comprise heterozygote transgenic cells.
  • a chimeric transgenic animal may comprise a cell having a genome that does not include a toolbox and a cell having a genome that contains at least one toolbox.
  • a chimeric transgenic animal can comprise a first cell and a second cell having a genome containing at least one toolbox.
  • the toolbox included in the genome of the first cell and the type of toolbox included in the genome of the second cell, the number of toolboxes, and the locus of the toolbox in the chimeric transgenic animal are the same as each other. Not.
  • a transgenic animal comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising the first toolbox
  • the genome of the first cell is included in the genome of the first cell.
  • the number of the first toolbox and the number of the first toolbox included in the genome of the second cell are different, or ii) the locus of the first toolbox and the genome of the second cell included in the genome of the first cell.
  • at least one or more of the positions of the first toolbox included in the different transgenic animals may be chimeric transgenic animals.
  • a transgenic animal comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox different from the first toolbox
  • the number of the first toolbox included in the genome of the first cell is equal to the number of the second toolbox included in the genome of the second cell
  • the first toolbox included in the genome of the first cell may be a chimeric transgenic animal.
  • a transgenic animal comprising a first cell having a genome comprising a first toolbox and a second cell having a genome comprising a second toolbox different from the first toolbox
  • the first cell The number of the first toolbox included in the genome of the second toolbox and the number of the second toolbox included in the genome of the second cell are different, or ii) the position of the first toolbox included in the genome of the first cell and the The transgenic animal may be a chimeric transgenic animal when at least one or more of the loci of the second toolbox included in the genome of the second cell are different.
  • Methods for producing a transgenic animal with a toolbox inserted therein include a method for producing a transgenic animal from animal cells, a method for producing a transgenic animal through delivery of a toolbox to an animal's tissue or organ, and transfection of the transgenic animal.
  • a method of making a converting animal Methods of making a transgenic animal from animal cells include a method of making a transgenic animal from wild-type animal cells and a method of making a transgenic animal from transgenic animal cells.
  • the transgenic animal produced by any of the above methods may be either a chimeric transgenic animal or a homologous transgenic animal.
  • Transgenic animals can be constructed, including the delivery of a toolbox to wild-type animal cells.
  • transgenic animals may be prepared through somatic cell transfer (hereinafter referred to as SCNT) after somatic cell microinjection of polynucleotides into wild type somatic cells.
  • SCNT somatic cell transfer
  • the transgenic animal may be a homologous transgenic animal.
  • transgenic animals can be produced by gamete microinjection into wild-type gametes.
  • the transgenic animal may be a chimeric transgenic animal.
  • transgenic animals can be made via zygote microinjection to wild-type conjugates.
  • the transgenic animal may be a chimeric transgenic animal.
  • transgenic animals can be produced in embryonic microinjection into wild-type embryos.
  • the transgenic animal may be a chimeric transgenic animal.
  • Transgenic animal cells can be used to construct transgenic animals.
  • transgenic somatic cells can be used to construct transgenic animals via SCNT.
  • the transgenic animal may be a homologous transgenic animal.
  • transgenic animals may be prepared through SCNT after somatic cell microinjection into transgenic somatic cells.
  • the transgenic animal may be a homologous transgenic animal.
  • transgenic animals can be produced by gamete microinjection into transgenic germ cells.
  • the transgenic animal may be a chimeric transgenic animal.
  • transgenic animals can be made via zygote microinjection to the transgenic conjugate.
  • the transgenic animal may be a chimeric transgenic animal.
  • transgenic animals can be prepared through embryo microinjection into transgenic embryos.
  • the transgenic animal may be a chimeric transgenic animal.
  • the toolbox delivery to animal tissues or organs can make the animal a transgenic animal.
  • a transgenic animal can be produced by microinjection into the animal's mammary tissue.
  • the transgenic animal may be a chimeric transgenic animal.
  • a transgenic animal can be produced by microinjection into an animal's reproductive organs.
  • the offspring obtained from the germ cells of the transgenic animal may also be a transgenic animal.
  • the transgenic animal may be a chimeric transgenic animal.
  • the crossover of the transgenic animal with the wild-type animal can produce a transgenic animal.
  • the transgenic animal may be produced by crossing the first transgenic animal and the second transgenic animal.
  • the second transgenic animal may be a progeny or blood-related of the first transgenic animal. Alternatively, the second transgenic animal may not be related to the first transgenic animal.
  • the transgenic animal obtained through the cross may include the same toolbox as some of the toolboxes included in the animal genome of the first transgenic animal at the same location.
  • the transgenic animal obtained through the cross may include the same toolbox as some of the toolboxes included in the animal genome of the second transgenic animal at the same location.
  • the transgenic animal obtained through the cross may be a homologous transgenic animal.
  • the transgenic animal obtained through the cross may include transgenic cells that are homozygous.
  • the transgenic animal obtained through the cross may include a transformed cell which is a heterozygote.
  • Transgenic animals with toolboxes can be used as breeders.
  • the breed improved animal may include, but is not limited to, a cow knocked out of a polynucleotide encoding beta-lactoglobulin and a cow knocked out of a polynucleotide encoding omega3.
  • the transgenic animal with the toolbox inserted can be used as a disease model animal.
  • the disease model animal may include, but is not limited to, a cow knocked out a polynucleotide encoding a tumor suppressor protein.
  • Transgenic animals with toolboxes can be used as disease resistant animals.
  • the disease resistant animal may include, but is not limited to, a cow knocked out a polynucleotide encoding a prion protein.
  • Organs, meat, skin, hair, and body fluids of the transgenic animal into which the toolbox is inserted may be used, but are not limited thereto.
  • the transgenic animal with the toolbox inserted can be used as a bioreactor.
  • the body fluids of the transgenic animals can be obtained.
  • the body fluid may comprise milk, blood, or urine.
  • Biomolecules can be obtained from the body fluids of the transgenic animals.
  • the biomolecule may comprise a protein.
  • the protein may comprise a protein of interest.
  • the transformed cell may comprise at least one toolbox on the animal genome. Any one of the toolboxes may include at least one RRS.
  • the toolbox may include one RRS.
  • the first end region of the toolbox may comprise one RRS.
  • the second terminal region of the toolbox may comprise one RRS.
  • the core area of the toolbox may include one RRS.
  • the toolbox may include more than one RRS.
  • the two or more RRS may include RRS 1 and RRS 2.
  • the RRS 1 may be located in any one of a first end region, a second end region, and a core region of the toolbox.
  • the RRS 2 may be located in any one of a first end region, a second end region, and a core region of the toolbox.
  • both RRS 1 and RRS 2 may be located in the core region.
  • RRS 1 may be located in the first end region and RRS 2 may be located in the second end region.
  • the RRS 1 and the RRS 2 may be identical to each other.
  • the RRS 1 and the RRS 2 may be located in the same direction.
  • the RRS 1 and the RRS 2 may be located in opposite directions.
  • the RRS 1 and the RRS 2 may be different from each other.
  • the RRS 1 and the RRS 2 may be located in the same direction.
  • the RRS 1 and the RRS 2 may be located in opposite directions.
  • the RRS 1 and the RRS 2 may be different from each other.
  • SSR 1 that can specifically interact with RRS 1 and SSR 2 that can specifically interact with RRS 2 may be the same or different from each other.
  • the RRS 1 and the RRS 2 may be different from each other.
  • the RRS 1 and RRS 2 may be paired with each other to cause interchange.
  • the RRS 1 and the RRS 2 do not form a pair so that interchange may not occur.
  • the intracellular animal genome may comprise polynucleotides encoding SSRs specific to any of the RRSs included in the toolbox.
  • the polynucleotides encoding the SSR may be included in a toolbox containing the RRS.
  • the polynucleotides encoding the SSRs can be included in other toolboxes.
  • the SSR may be provided when no polynucleotide encoding an SSR capable of interacting with any of the RRSs included in the toolbox is present on the animal genome in the cell.
  • the form that provides the SSR is a naked nucleic acid vector, a non-viral vector, a viral vector, or a recombinase comprising a polynucleotide encoding the SSR. It may be a polypeptide (recombinase polypeptide).
  • the toolbox may include at least one RRS.
  • the cell comprising the toolbox may be provided with an exo-polynucleotide comprising an RRS paired with any one of the RRSs included in the toolbox.
  • insertion of the exo-polynucleotide into the toolbox may occur through interaction with SSR specific to the pair of RRSs.
  • the toolbox may include more than one RRS.
  • the two or more RRS may include RRS 1 and RRS 2.
  • the RRS 1 and the RRS 2 form a pair and may be located in the same direction.
  • two loxps may be located in the same direction in the core area of the toolbox.
  • removal of polynucleotides located between the pair of RRSs may occur through interaction with SSRs specific to the pair of RRSs.
  • SSRs specific to the pair of RRSs For example, if the core region of the toolbox contains a polynucleotide encoding a gRNA between two loxps, Cre recombinase may be provided to remove the polynucleotide encoding the gRNA from the toolbox.
  • the toolbox may include more than one RRS.
  • the two or more RRS may include RRS 1 and RRS 2.
  • the RRS 1 and the RRS 2 may be paired and positioned in opposite directions.
  • inversion of polynucleotides located between the pair of RRSs may occur through interaction with SSRs specific to the pair of RRSs.
  • the toolbox may include more than one RRS.
  • the two or more RRS may include RRS 1 and RRS 2.
  • the RRS 1 and the RRS 2 may not be paired.
  • the core region of the toolbox may include loxp (RRS 1) and loxp2722 (RRS 2).
  • the loxp may be located upstream of loxp2722.
  • Cells containing the toolbox may be provided with exo-polynucleotides comprising RRS 3 paired with RRS 1 and RRS 4 paired with RRS 2.
  • the exo-polynucleotides may include loxp (RRS 3) and loxp2722 (RRS 4).
  • the loxp may be located upstream of loxp2722.
  • interaction with SSR specific to RRS 1 and RRS 3 and interaction with SSR specific to RRS 2 and RRS 4 may occur.
  • replacement of a polynucleotide located between RRS 1 and RRS 2 and a polynucleotide located between RRS 3 and RRS 4 may occur.
  • Non-transcribed polynucleotides located within and polynucleotides encoding gRNAs of exo-polynucleotides can be exchanged.
  • the toolbox may include more than one RRS.
  • the two or more RRS may include RRS 1 and RRS 2.
  • the RRS 1 and the RRS 2 may not be paired.
  • the core region of the toolbox may include loxp (RRS 1) and loxp2722 (RRS 2).
  • the loxp may be located upstream of loxp2722.
  • Cells comprising the toolbox may be provided with a first exo-polynucleotide comprising RRS 3 paired with RRS 1 and a second exo-polynucleotide comprising RRS 4 paired with RRS 2.
  • the first exo-polynucleotide may comprise loxp (RRS 3), and the second exo-polynucleotide may comprise loxp2722 (RRS 4).
  • the first exo-polynucleotide may be inserted at the RRS 1 position, and the second exo-polynucleotide may be inserted at the RRS 2 position. That is, two or more kinds of exo-polynucleotides may be inserted into a cell having an animal genome including a toolbox including two or more RRS.
  • two or more types of exo-polynucleotides in the toolbox may be inserted as desired, respectively, as well as two or more types of exo-polynucleotides already present in the toolbox may be removed or replaced.
  • the toolbox may include loxp, loxp mutant, rox, and attP.
  • a cell including the toolbox may be provided with a first exo-polynucleotide consisting of a rox variant and Cas9 paired with rox included in the foil gas.
  • insertion of the first exo-polynucleotide into the toolbox may occur through interaction with Dre specific for the rox and rox variants.
  • a cell comprising the toolbox includes a rox variant paired with rox included in the toolbox, a first exo-polynucleotide composed of Cas9, and a second pair composed of attB and gRNA paired with attP included in the toolbox.
  • Exo-polynucleotides may be provided.
  • the insertion of the first exo-polynucleotide and the second exo-polynucleotide into the toolbox may occur through interaction with Dre specific to the rox and rox variants and interaction with PhiC31 specific to the attP and attB. have.
  • Cas9 and gRNA can be expressed at the same time in the cell including the toolbox, so that the CRISPR / enzyme system can be operated even if Cas9 or gRNA is not delivered separately.
  • Second exo-polynucleotides present between the mutants may be provided. In this case, insertion of the first exo-polynucleotide into the toolbox through interaction with Dre specific to the rox and rox variants and Cre to the loxp and loxp variants occurs and the second exo-polynucleotide Replacement may take place.
  • location-specific recombination may occur in the toolbox located at the desired locus. have.
  • a transformed cell having an animal genome comprising a first toolbox comprising RRS1 and a second toolbox comprising RRS2 may be provided with an exo-polynucleotide comprising an RRS1 or RRS1 variant paired with RRS1.
  • insertion of the exo-polynucleotide into the first toolbox may occur through interaction of the RRS1 or RRS1 variant with specific SSR1.
  • a transgenic cell having an animal genome comprising a first toolbox including RRS1 and RRS2 and a second toolbox including RRS1 and RRS3 is combined with RRS1 or RRS1 variants and RRS2 paired with RRS1.
  • Exo-polynucleotides located between paired RRS2 or RRS2 variants can be provided.
  • the polynucleotide located between RRS1 and RRS2 of the first toolbox and RRS1 or RRS1 variant and RRS2 through interaction of the RRS1 or RRS1 variant with specific SSR1 and the RRS2 or RRS2 variant with specific SSR2 Or replacement of exo-polynucleotides located between RRS2 variants may occur.
  • an exo comprising an RRS1 or RRS1 variant paired with RRS1 in a transformed cell having an animal genome comprising a first toolbox comprising two or more RRS1 and a second toolbox comprising RRS2 Polynucleotides may be provided.
  • the RRS1 or RRS1 variant may interact with SSR1 specific to the RRS1 or RRS1 variant.
  • SSR1 specific to the RRS1 or RRS1 variant.
  • the transformed cell may comprise at least one toolbox. Any one of the at least one toolbox may include at least one RRS. Transformation through site-specific recombination is possible using the at least one RRS.
  • the transformed cell may comprise an edited toolbox.
  • Edited toolbox means a toolbox in which the location-specific recombination has occurred.
  • Transgenic cells comprising at least one edited toolbox may be diploid cells.
  • the diploid cell is as previously mentioned.
  • Transgenic cells comprising at least one edited toolbox may be haploid cells.
  • the haploid cells are as mentioned above.
  • Transgenic cells comprising two or more edited toolboxes may be homozygote.
  • Transgenic cells comprising two or more edited toolboxes may be heterozygote.
  • Transgenic cells comprising at least one edited toolbox may form cell colonies.
  • Homologous cell colonies have the same toolbox that each cell contains and the edited toolbox that each cell contains.
  • Chimeric cell colonies refer to cell colonies other than homologous cell colonies.
  • the edited toolbox may contain polynucleotides encoding fluorescent proteins.
  • an exo-polynucleotide encoding a fluorescent protein using site-specific recombination can be inserted into a toolbox comprising at least one RRS.
  • animal cells comprising the edited toolbox can be distinguished from animal cells not containing the edited toolbox.
  • the edited toolbox may contain an antibiotic resistance gene.
  • the animal genome may comprise the edited toolbox above.
  • an exo-polynucleotide encoding an antibiotic resistance gene may be inserted using a site-specific recombination in a toolbox comprising at least one RRS.
  • Animal cells comprising the edited toolbox can survive treatment with antibiotics.
  • animal cells comprising the edited toolbox can be separated from animal cells not containing the edited toolbox.
  • the edited toolbox may comprise a polynucleotide encoding an antigen or a nucleotide that can act as an antigen.
  • Animal cells may comprise the edited toolbox.
  • an exo-polynucleotide comprising a nucleotide capable of acting as an antigen using site-specific recombination may be inserted into a toolbox comprising at least one RRS.
  • Animal cells comprising the edited toolbox may interact with antibodies specific for the antigen. Thus, animal cells comprising the edited toolbox can be distinguished from animal cells not containing the edited toolbox.
  • the edited toolbox may include polynucleotides that encode surface markers.
  • Animal cells may comprise the edited toolbox.
  • polynucleotides encoding surface markers using site-specific recombination can be inserted into a toolbox comprising at least one RRS.
  • Animal cells comprising the edited toolbox can interact with antibodies specific for the surface markers.
  • the antibody may interact with magnetic particles or fluorophores. Therefore, the animal cells including the edited toolbox can be distinguished from animal cells not containing the edited toolbox through magnetic properties or fluorescence signals.
  • the toolbox may comprise a polynucleotide encoding a suicide gene.
  • the edited toolbox may not include a polynucleotide encoding the suicide gene.
  • the core region of the toolbox may include loxp, suicide gene, and loxp variants in order.
  • exo-polynucleotides containing loxp at the 5 'end and loxp variants at the 3' end may be replaced with the suicide gene through site-specific recombination.
  • Animal cells containing the edited toolbox do not contain suicide genes and thus do not produce apoptosis even when a prodrug is provided. Thus, animal cells comprising the edited toolbox can be distinguished from animal cells not containing the edited toolbox.
  • Each cell included in a homologous transgenic animal may comprise at least one toolbox.
  • the at least one toolbox may include at least one edited toolbox.
  • Chimeric transgenic animals means transgenic animals other than homologous transgenic animals.
  • Methods of making a transgenic animal inserted with an edited toolbox include a method of making from an animal cell, a method of making it through exo-polynucleotide transfer or SSR introduction into an animal tissue or organ, and mating of a transgenic animal.
  • Methods for making from animal cells include methods for making from transgenic animal cells into which a toolbox comprising at least one RRS is inserted.
  • the transgenic animal produced by any of the above methods may be either a chimeric transgenic animal or a homologous transgenic animal.
  • a transgenic animal inserted with an edited toolbox can be produced from a transgenic animal cell into which a toolbox including at least one RRS is inserted.
  • transgenic animals may be prepared through SCNT after introducing SSR into somatic cells into which a toolbox including at least one RRS is inserted.
  • a transgenic animal may be manufactured through SCNT after somatic cell microinjection of SSR and polynucleotide comprising at least one RRS in somatic cells into which a toolbox including at least one RRS is inserted.
  • the transgenic animal may be a homologous transgenic animal.
  • a transgenic animal may be produced by gamete microinjection of polynucleotides containing SSR into germ cells into which a toolbox including at least one RRS is inserted.
  • a transgenic animal may be prepared by gamete microinjection of polynucleotides including SSR and polynucleotides including at least one RRS in germ cells into which a toolbox including at least one RRS is inserted. have.
  • the transgenic animal may be a chimeric transgenic animal.
  • a transgenic animal can be prepared by introducing an SSR into a conjugate into which a toolbox including at least one RRS is inserted.
  • a transgenic animal may be prepared by zygote microinjection of a polynucleotide comprising at least one RRS while treating an SSR to a conjugate into which a toolbox including at least one RRS is inserted.
  • the transgenic animal may be a chimeric transgenic animal.
  • a transgenic animal can be prepared by introducing SSR into an embryo into which a toolbox including at least one RRS is inserted.
  • a transgenic animal may be prepared by embryo microinjection of a polynucleotide encoding SSR and a polynucleotide comprising at least one RRS into an embryo into which a toolbox including at least one RRS is inserted.
  • the transgenic animal may be a chimeric transgenic animal.
  • the tissue or organ of the animal may comprise a cell into which a toolbox comprising at least one RRS is inserted.
  • Transgenic animals can be constructed that include an edited toolbox by introducing SSR into the tissue or organ of the animal or by delivery of exo-polynucleotides.
  • a transgenic animal comprising a toolbox edited by microinjecting a polynucleotide encoding SSR and a polynucleotide comprising at least one RRS into the mammary gland tissue may be produced.
  • the transgenic animal may be a chimeric transgenic animal.
  • a transgenic animal inserted with an edited toolbox may be prepared by micro-injecting SSR into the reproductive organ of the animal.
  • the offspring obtained from the germ cells of the transgenic animal may also be a transgenic animal.
  • the transgenic animal may be a chimeric transgenic animal.
  • the transgenic animal can be produced by crossing the first transgenic animal and the second transgenic animal.
  • a trait comprising a toolbox edited by crossing a first transgenic animal inserted with a toolbox including at least one RRS and a second transgenic animal inserted with a toolbox including a polynucleotide encoding an SSR.
  • Transgenic animals can be produced.
  • the second transgenic animal may be a progeny or blood-related of the first transgenic animal. Alternatively, the second transgenic animal may not be related to the first transgenic animal.
  • the transgenic animal obtained through the cross may include the same toolbox as some of the toolboxes included in the animal genome of the first transgenic animal at the same location.
  • the transgenic animal obtained through the cross may include the same toolbox as some of the toolboxes included in the animal genome of the second transgenic animal at the same location.
  • the transgenic animal obtained through the cross may be a homologous transgenic animal.
  • the transgenic animal obtained through the cross may include transgenic cells that are homozygous.
  • the transgenic animal obtained through the cross may include a transformed cell which is a heterozygote.
  • Transgenic animals with an edited toolbox can be used as breeders.
  • the transgenic animal with the edited toolbox inserted can be used as a disease model animal.
  • Transgenic animals inserted with an edited toolbox can be used as disease resistant animals.
  • Organs, meat, skin, hair, and body fluids of the transgenic animal into which the edited toolbox is inserted may be used, but are not limited thereto.
  • Transgenic animals inserted with an edited toolbox can be used as bioreactors.
  • RNA-guide endonucleases and guide nucleic acids can be introduced for site-specific transformation on an animal genome including a toolbox.
  • the RNA-guide endonuclease may comprise Cas9, but is not limited thereto.
  • the guide nucleic acid may include gRNA, but is not limited thereto.
  • the Cas9 and gRNA can be introduced in separate forms into cells comprising the toolbox.
  • Forms that provide Cas9 may include DNA plasmids, DNA linear fragments (DNA linear fragments), RNA linear fragments (RNA linear fragments), and proteins.
  • the RNA linear fragment may include mRNA of Cas9.
  • Forms that provide the gRNA can include DNA plasmids, DNA linear fragments, and RNA linear fragments.
  • Forms that provide the Cas9 and gRNA together may comprise a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • Cas9 and gRNA within the cell containing the toolbox may be provided as one delivery vector.
  • Forms providing the Cas9 and gRNA may comprise one DNA linear fragment (DNA linear fragment), and RNA linear fragment (RNA linear fragment).
  • the toolbox included in the animal genome may include at least one of an RNA-guide endonuclease and a guide nucleic acid.
  • the cells in which the site-specific transformation will take place can express some or all of the components of the crisper / enzyme system so that the site-specific transformation can be performed conveniently.
  • the toolbox may comprise at least one polynucleotide encoding an RNA-guide endonuclease.
  • the RNA-guided endonuclease may include, but is not limited to, Cas9 or Cas9 mutant.
  • the toolbox may comprise one or more polynucleotides encoding Cas9.
  • the toolbox may comprise a promoter capable of initiating transcription of the Cas9.
  • the promoter may be any one of a constitutive promoter, a tissue-specific promoter, and an inducible promoter.
  • the toolbox may comprise one or more polynucleotides encoding Cas9.
  • the toolbox may comprise a promoter capable of initiating transcription of the Cas9.
  • the toolbox may include one or more RRS.
  • the toolbox may comprise one or more RRS at the 5 ′ end of a promoter capable of initiating transcription of the Cas9.
  • the toolbox may comprise one or more RRS between a promoter capable of initiating transcription of the Cas9 and a polynucleotide encoding Cas9.
  • the toolbox may comprise an RRS at the 3 ′ end of the polynucleotide encoding the Cas9.
  • the toolbox may include at least one RRS at the 5 ′ end of the polynucleotide encoding the Cas9 and at least one RRS at the 3 ′ end of the polynucleotide encoding the Cas9.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may interact with the same SSR.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may be the same as each other.
  • the toolbox may comprise at least one polynucleotide encoding a guide nucleic acid.
  • the guide nucleic acid may include gRNA, but is not limited thereto.
  • the toolbox may comprise one or more polynucleotides encoding gRNAs.
  • the toolbox may comprise a promoter capable of initiating transcription of gRNAs.
  • the promoter can be any one of a constitutive expression promoter, a tissue-specific promoter, and an inducible promoter.
  • the constitutive expression promoter capable of initiating transcription of the gRNA may include, but is not limited to, a U6 promoter.
  • the toolbox may comprise one or more polynucleotides encoding gRNAs.
  • the toolbox may comprise a promoter capable of initiating transcription of gRNAs.
  • the toolbox may include one or more RRS.
  • the toolbox may comprise one or more RRS at the 5 ′ end of a promoter capable of initiating transcription of the gRNA.
  • the toolbox may comprise one or more RRS between a promoter capable of initiating transcription of the gRNA and a polynucleotide encoding a gRNA.
  • the toolbox may comprise an RRS at the 3 ′ end of the polynucleotide encoding the gRNA.
  • the toolbox may include one or more RRS at the 5 ′ end of the polynucleotide encoding the gRNA, and one or more RRS at the 3 ′ end of the polynucleotide encoding the gRNA.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may interact with the same SSR.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may be the same as each other.
  • the toolbox may comprise at least one polynucleotide encoding an RNA-guide endonuclease and at least one polynucleotide encoding a guide nucleic acid.
  • the RNA-guide endonuclease may comprise Cas9, but is not limited thereto.
  • the guide nucleic acid may include gRNA, but is not limited thereto.
  • the toolbox may comprise one or more polynucleotides encoding Cas9.
  • the toolbox may comprise a promoter capable of initiating transcription of the Cas9.
  • the promoter can be any one of a constitutive expression promoter, a tissue-specific promoter, and an inducible promoter.
  • the toolbox may comprise one or more polynucleotides encoding gRNAs.
  • the toolbox may comprise a promoter capable of initiating transcription of gRNAs.
  • the promoter can be any one of a constitutive expression promoter, a tissue-specific promoter, and an inducible promoter.
  • the constitutive expression promoter capable of initiating transcription of the gRNA may include, but is not limited to, a U6 promoter.
  • the toolbox may comprise one or more polynucleotides encoding Cas9.
  • the toolbox may comprise a promoter capable of initiating transcription of the Cas9.
  • the toolbox may comprise one or more polynucleotides encoding gRNAs.
  • the toolbox may comprise a promoter capable of initiating transcription of gRNAs.
  • the toolbox may include one or more RRS.
  • the toolbox may comprise one or more RRS at the 5 ′ end of a promoter capable of initiating transcription of the Cas9.
  • the toolbox may comprise one or more RRS between a promoter capable of initiating transcription of the Cas9 and a polynucleotide encoding Cas9.
  • the toolbox may comprise an RRS at the 3 ′ end of the polynucleotide encoding the Cas9.
  • the toolbox may include at least one RRS at the 5 ′ end of the polynucleotide encoding the Cas9 and at least one RRS at the 3 ′ end of the polynucleotide encoding the Cas9.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may interact with the same SSR.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may be the same as each other.
  • the toolbox may comprise one or more RRS at the 5 ′ end of a promoter capable of initiating transcription of the gRNA.
  • the toolbox may comprise one or more RRS between a promoter capable of initiating transcription of the gRNA and a polynucleotide encoding a gRNA.
  • the toolbox may comprise an RRS at the 3 ′ end of the polynucleotide encoding the gRNA.
  • the toolbox may include one or more RRS at the 5 ′ end of the polynucleotide encoding the gRNA, and one or more RRS at the 3 ′ end of the polynucleotide encoding the gRNA.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may interact with the same SSR.
  • any one of the 5 'terminal RRS and the 3' terminal RRS may be the same as each other.
  • the toolbox may comprise at least one polynucleotide encoding an RNA-guide endonuclease.
  • the RNA-guide endonuclease may comprise Cas9, but is not limited thereto.
  • the toolbox may comprise a polynucleotide encoding Cas9.
  • the toolbox may comprise a promoter that initiates the transcription of a polynucleotide encoding Cas9.
  • the promoter may comprise a tissue-specific promoter or an inducible promoter.
  • polynucleotides encoding Cas9 included in the toolbox may be transcriptionally initiated when the toolbox is included in a specific tissue cell.
  • tissue-specific promoter is a mammary tissue specific promoter
  • polynucleotides encoding Cas9 included in the toolbox may be transcriptionally initiated when the toolbox is included in mammary tissue cells.
  • the mammary tissue specific promoters include alpha-casein promoter, beta-casein promoter, kappa-casein promoter, and mu-casein promoter. , And beta-lactoglobulin promoters, but are not limited thereto.
  • tissue-specific promoter is a reproductive organ specific promoter
  • polynucleotides encoding Cas9 included in the toolbox may be transcriptionally initiated when the toolbox is included in germ cells.
  • the reproductive organ specific promoter may include, but is not limited to, an ovarian-specific promoter and a testis-specific promoter.
  • the promoter initiating transcription of a polynucleotide encoding Cas9 is a tissue-specific promoter, it is possible to limit where site-specific transformation occurs in the transgenic animal comprising the toolbox. In addition, unnecessary site-specific transformation can be prevented from occurring in other tissues of the transgenic animal.
  • the promoter is an inducible promoter, transcription can be initiated when certain conditions are met.
  • the induction promoter may include, but is not limited to, a chemically inducible promoter, a temperature inducible promoter, and a light inducible promoter.
  • Chemically inducible promoters can initiate transcription in the presence of certain chemical compounds.
  • the chemically inducible promoters include antibiotic-inducible promoters, alcohol-inducible promoters, steroid-inducible promoters, and metal-induced promoters. metal-inducible promoter), but is not limited thereto.
  • the antibiotic-inducible promoter may include, but is not limited to, a Tet-on promoter and a Tet-off promoter.
  • the steroid-inducible promoter includes, but is not limited to, an estrogen-inducible promoter.
  • the metal-inducible promoter may include a copper-inducible promoter, but is not limited thereto.
  • Temperature inducible promoters can initiate transcription if temperature conditions are met.
  • the temperature inducible promoter may include a heat shock-inducible promoter and a cold shock-inducible promoter, but is not limited thereto.
  • the heat shock-inducible promoter may include an Hsp promoter, but is not limited thereto.
  • the light inducible promoter may initiate transcription if the wavelength condition of the light is satisfied.
  • the promoter for initiating transcription of a polynucleotide encoding Cas9 is an inducible promoter, it is possible to control when the site-specific transformation occurs in a transgenic cell or animal comprising the toolbox.
  • the toolbox may comprise a polynucleotide encoding Cas9.
  • the toolbox may comprise an RRS at the 5 ′ end of the polynucleotide encoding the Cas9.
  • Nucleic acid may include a polynucleotide encoding a promoter.
  • the promoter may comprise a constitutive expression promoter, a tissue-specific promoter, or an inducible promoter.
  • the nucleic acid may include an RRS at one or both ends of a polynucleotide encoding a promoter. The RRS may be paired with an RRS included in the toolbox.
  • the toolbox may provide an SSR capable of interacting with the nucleic acid and the RRS. Insertion of a polynucleotide encoding the promoter into the toolbox may occur.
  • the polynucleotide encoding Cas9 may not be transcriptionally initiated prior to insertion of the polynucleotide encoding the promoter and may be transcriptionally initiated after insertion.

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Abstract

La présente invention concerne un animal transgénique et un embryon transgénique, produisant un élément constitutif d'une nucléase artificielle. L'animal transgénique (ou embryon) produisant un élément constitutif d'une nucléase artificielle selon l'invention comprend : une première cellule ayant un génome comprenant une première boîte à outils; et une seconde cellule ayant un génome comprenant une seconde boîte à outils, la première boîte à outils et la seconde boîte à outils comprenant chacune au moins l'un d'un polynucléotide codant pour une endonucléase guidée par ARN, ou un polynucléotide codant pour un acide nucléique de guidage capable de se lier spécifiquement à un site cible, et la première boîte à outils étant située au niveau d'un premier locus du génome de la première cellule, la seconde boîte à outils étant située au niveau d'un second locus du génome de la seconde cellule, et le premier locus et le second locus étant différents l'un de l'autre.
PCT/KR2019/010388 2018-08-16 2019-08-14 Animal transgénique et embryon transgénique produisant une nucléase artificielle Ceased WO2020036445A1 (fr)

Priority Applications (9)

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BR112020000388-3A BR112020000388B1 (pt) 2018-08-16 2019-08-14 Método para produzir um embrião transgênico não humano tendo um genoma incluindo um gene com um sítio modificado
AU2019275586A AU2019275586B2 (en) 2018-08-16 2019-08-14 Transgenic animals and transgenic embryos producing an engineered nuclease
US16/626,227 US12029204B2 (en) 2018-08-16 2019-08-14 Transgenic animals and transgenic embryos producing an engineered nuclease
JP2019562368A JP7060256B2 (ja) 2018-08-16 2019-08-14 改変ヌクレアーゼを産生するトランスジェニック動物およびトランスジェニック胚
AU2021232802A AU2021232802B2 (en) 2018-08-16 2021-09-17 Transgenic animals and transgenic embryos producing an engineered nuclease
AU2021232801A AU2021232801B2 (en) 2018-08-16 2021-09-17 Transgenic animals and transgenic embryos producing an engineered nuclease
JP2022063839A JP7430414B2 (ja) 2018-08-16 2022-04-07 改変ヌクレアーゼを産生するトランスジェニック動物およびトランスジェニック胚
JP2024008570A JP7671946B2 (ja) 2018-08-16 2024-01-24 改変ヌクレアーゼを産生するトランスジェニック動物およびトランスジェニック胚
US18/593,618 US20240298617A1 (en) 2018-08-16 2024-03-01 Transgenic animals and transgenic embryos producing an engineered nuclease

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US201862764905P 2018-08-16 2018-08-16
US62/764,905 2018-08-16
KR1020190065613A KR102103103B1 (ko) 2018-08-16 2019-06-03 인위적 뉴클레아제를 생산하는 형질전환 동물 및 형질전환 배아
KR10-2019-0065613 2019-06-03

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