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WO2019222974A1 - Procédé d'inactivation du gène de souris ebln1 en utilisant le système crispr/cas9 - Google Patents

Procédé d'inactivation du gène de souris ebln1 en utilisant le système crispr/cas9 Download PDF

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Publication number
WO2019222974A1
WO2019222974A1 PCT/CN2018/088286 CN2018088286W WO2019222974A1 WO 2019222974 A1 WO2019222974 A1 WO 2019222974A1 CN 2018088286 W CN2018088286 W CN 2018088286W WO 2019222974 A1 WO2019222974 A1 WO 2019222974A1
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WIPO (PCT)
Prior art keywords
ebln1
gene
grna
cas9
crispr
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Ceased
Application number
PCT/CN2018/088286
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English (en)
Chinese (zh)
Inventor
毛吉炎
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Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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Priority to PCT/CN2018/088286 priority Critical patent/WO2019222974A1/fr
Publication of WO2019222974A1 publication Critical patent/WO2019222974A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • 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
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

  • the present invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a method for knocking out mouse EBLN1 gene by using CRISPR / Cas9 system.
  • Borna disease virus is a non-segment single-stranded negative-stranded RNA virus first discovered in Germany in 1885. It has strict neuropathy, and its genome encodes six viral proteins. Nucleoprotein (N) is the main infection marker and pathogenic protein. Studies have shown that similar fragments to the Bornavirus N gene (encoding nucleoprotein) are found in a variety of mammalian genomes, including humans, non-human primates, rodents, and elephants, and are named endogenous Endogenous Boma-like N Element (EBLN).
  • EBLN Endogenous Endogenous Boma-like N Element
  • the EBLN1 gene may be a potential susceptible gene that causes depression, which encodes a protein or participates in the pathophysiology of depression, and conducts in-depth systematic research on it, and further clarifies the molecular genetic basis of depression It is of great significance to search for drug treatment targets, but the lack of E BLN1 gene knockout mice in the prior art has caused certain obstacles to the progress of related research.
  • the present invention provides a method for using a CRISPR / Cas9 system to knock out mouse EBLN 1 gene.
  • the technical solution adopted by the present invention is: A method for knocking out mouse EBLN1 gene by using CRISPR / Cas9 system, which is characterized in that it includes a sequence specific to a mouse EBLN1 gene target site The base sequence is shown in FIG. 1; the gRNA oligonucleotide sequence that specifically targets the EBLN1 gene is shown in FIG. 2.
  • An in vitro transcription vector comprising the gRNA.
  • the in vitro transcription vector of gRNA is pMD19T-T7-gRNA
  • the in vitro transcription vector of Cas9 protein is p ST1374-NLS-flag-linker-Cas9.
  • a method for knocking out a mouse EBLN1 gene using the CRISPR / Cas9 system includes the following steps
  • the gRNA and Cas9 mRNA of the above steps are injected into the cytoplasm of mouse fertilized eggs, cultured in vitro and transplanted into the fallopian tubes of adult female mice for generating transgenic mice containing EBLN1 gene mutations.
  • step S3 After gRNA and Cas9 mRNA are mixed in step S3, the final concentration is: Cas9 mRNA 30
  • the mRNA is transfected into the cell at the same time, and the EBLN1 gene is knocked out for studying the function of the EBLN1 gene;
  • gRNA and Cas9 mRNA are injected into a fertilized egg at the same time, and then used to produce transgenic mice that knock out EBLN 1 by embryo transfer;
  • FIG. 1 is a sequence diagram of a mouse EBLN1 gene-specific target site
  • FIG. 2 is a gRNA sequence diagram designed to target the EBLN1 gene.
  • FIG. 1 is a sequence diagram of a mouse EBLN1 gene-specific target site
  • FIG. 2 is a gRNA sequence diagram designed to target the EBLN 1 gene.
  • Invention Examples
  • the sequence of the mouse EBLN1 gene was found in GenBank and the gRNA was designed.
  • the target site sequence is shown in FIG. 1 and the gRNA sequence is shown in FIG. 2.
  • I site was digested, ligated, and inserted into pMD19T-T7-gRNA.
  • the CRISPR / Cas9 system for the EBLN1 gene is:
  • the in vitro transcription vector pST1374-NLS-flaglinker-Cas9 performs in vitro transcription mediated by the T7 promoter.
  • pST1374-NLS-flag-linker-Cas9 was linearized with Age I; then TMD in vitro transcription kit (Biomax) was used to perform pMD19T-T7-gRNA, pST1374-NLS-flag-linker-Cas9 Transcription; Finally, MEGA Clear kit (Ambion) was used to purify gRNA and Cas9 mRNA.
  • Example 3 Application of CRISPR / Cas9 system targeting mouse EBLN1 gene to generate transgenic mice containing EBLN1 gene mutation
  • the embryos at the single-cell stage were collected in a naturally mated donor female mouse by surgery, and the premixed gRNA and Cas9 mRNA (the final concentration after mixing was Cas9 mRNA 30 using a microinjector).
  • ng / ⁇ il gRNA 12 ng / ⁇ il
  • gRNA 12 ng / ⁇ il is injected into the cytoplasm of fertilized eggs.
  • the injected fertilized eggs were transferred to Quinn's Advantage Cleavage Medium, cultured in vitro at 37 ° C for 24 hours, and then transplanted into recipient female mice to generate transgenic mice containing EBLN1 gene mutations.
  • the mRNA is transfected into the cell at the same time, and the EBLN1 gene is knocked out for studying the function of the EBLN1 gene;
  • gRNA and Cas9 mRNA are injected into a fertilized egg at the same time, and then used to produce transgenic mice that knock out EBLN 1 by embryo transfer;
  • gRNA and Cas9 mRNA are transfected into cells at the same time, and positive cells are used as donor cells after screening

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé d'inactivation du gène de souris EBLN1 en utilisant un système CRISPR/Cas9. Le procédé comprend : premièrement, l'obtention de la séquence d'ADN pour la région de reconnaissance de l'ARNg du gène de souris EBLN1; puis, la conception des ARNg qui ciblent la séquence d'ADN et la construction d'un ARNg contenant le promoteur-T7 dans le vecteur de transcription in vitro pour le gène EBLN1; et enfin, l'utilisation de Cas9 et de l'ARNg dans le vecteur de transcription in vitro pour obtenir l'ARNm Cas9 et l'ARNg, ces derniers pouvant être utilisés pour produire la souris transgénique avec l'inactivation du gène EBLN1.
PCT/CN2018/088286 2018-05-24 2018-05-24 Procédé d'inactivation du gène de souris ebln1 en utilisant le système crispr/cas9 Ceased WO2019222974A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088286 WO2019222974A1 (fr) 2018-05-24 2018-05-24 Procédé d'inactivation du gène de souris ebln1 en utilisant le système crispr/cas9

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088286 WO2019222974A1 (fr) 2018-05-24 2018-05-24 Procédé d'inactivation du gène de souris ebln1 en utilisant le système crispr/cas9

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WO2019222974A1 true WO2019222974A1 (fr) 2019-11-28

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103160514A (zh) * 2013-03-13 2013-06-19 重庆医科大学 人EBLN-1基因cDNA全长核苷酸序列及其克隆方法
WO2016168840A1 (fr) * 2015-04-17 2016-10-20 University Of South Florida Modèle animal pour l'étude de maladies humaines complexes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103160514A (zh) * 2013-03-13 2013-06-19 重庆医科大学 人EBLN-1基因cDNA全长核苷酸序列及其克隆方法
WO2016168840A1 (fr) * 2015-04-17 2016-10-20 University Of South Florida Modèle animal pour l'étude de maladies humaines complexes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PENG HE ET AL.: "Knock-Down of Endogenous Bornavirus-Like Nucleoprotein 1 Inhibits Cell Growth and Induces Apoptosis in Human Oligodendroglia Cells", INT J MOL SCI., vol. 17, no. 4, 24 March 2016 (2016-03-24), XP055656716, ISSN: 1422-0067 *
ZHANG, LIANG: "Preliminary Study on Functions, Mechanisms of Human EBLNlGene and Its Role in Major Depressive Disorder", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, MEDICINE AND HEALTH SCIENCES, 15 July 2015 (2015-07-15), ISSN: 1674-022X *

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