RU2495126C2 - Recombinant plasmid pol-dsred2, coding oct4 and lin28 human proteins and fluorescent protein dsred2, designed to produce induced pluripotent human stem cells - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: plasmid genetic structure pOL-DsRed2 is produced, being built on the basis of a plasmid vector pIRES (Clontech), where fragments of cDNA of human genes OCT4 and LIN28 are placed, being connected with a nucleotide sequence coding P2A-peptide and gene cDNA, coding fluorescent protein DsRed2.

EFFECT: invention provides for simultaneous translation of human proteins OCT4 and LIN28 and fluorescent protein DsRed2 in production of induced pluripotent stem cells of a human being and animals in medicine and veterinary science.

3 cl, 1 dwg, 1 tbl

Description

The invention relates to the field of genetic engineering, molecular and cellular biology, biotechnology and can be used to produce induced pluripotent stem cells in humans and animals.

Induced pluripotent stem cells (iPSCs) are a type of stem cells that can be obtained from somatic cells of animals and humans as a result of increased expression of a set of specific genes. Overexpression of genes such as OCT4, SOX2, KLF4, and c-MYC has been successfully used to obtain human and animal iPSCs. Later, the Thomson group of human iPSCs were successfully obtained using the OCT4, SOX2, NANOG and LIN28 genes.

It is known that to obtain the majority of new iPSC lines, genetic constructs based on retro- and lentiviral vectors are currently used. This method is characterized by the fact that random copying of DNA copies of the genomes of retro- or lentiviruses into the cell genomes occurs, which in turn can lead to disruption of the functioning of genes.

For the full use of induced pluripotent stem cells in basic and applied research, such as screening for new drugs, studies in the field of toxicology and regenerative medicine, a number of problems need to be addressed. First, the development of methods for obtaining iPSCs without the genetic modification of their genomes is necessary. Secondly, the method of obtaining iPSCs should be quite effective.

It is known that plasmid vectors — plasmids — can temporarily exist in the nuclei of cells, providing stable transcription of nucleotide sequences controlled by constitutive promoters. In addition, a method based on the use of specific sequences — 2A peptides — is known that allows one to obtain genetic constructs that ensure translation of several polypeptides (proteins) from one molecule of messenger RNA [5].

The objective of the invention is the construction of a polycistronic non-integrating plasmid construct encoding human OCT4 and LIN28 proteins and the fluorescent protein DsRed2, which provides simultaneous translation of these proteins and is a vector for the production of induced pluripotent stem cells. Proteins OCT4 and LIN28 are necessary for triggering cell reprogramming, and the fluorescent protein DsRed2 serves as a marker for cell transfection and subsequent elimination of plasmid DNA.

The implementation of the invention is as follows.

The construction of recombinant plasmid DNA is performed on the basis of the plasmid pIRES (Clontech) and includes the following steps:

1. isolate RNA from cells of the known human embryonic stem cell line HUES9 and synthesize cDNA using oligo- (dT) primers. The obtained cDNA is used as a matrix for the synthesis of fragments encoding proteins;

2. synthesis of the cDNA fragment of the OCT4 gene - position in mRNA 53-1135- (Homo sapiens POU domein, class 5, Transcription factor 1 (POU5F1)), (registration number in GeneBank NM_002701), 1152 pairs of nucleotides containing 3 'in size -terminal sequence of the F2A peptide (OCT4-F2A). Synthesis was carried out by polymerase chain reaction with primers: hOCT45'NheI 5'-TTTTGCGCTAGCCATGGCGGGACACCTGGCTTCGG-3 '(forward primer contains an artificially introduced restriction endonuclease site Nhe I) and hOCT4 F2A 3'5'-CCTGCAAGTTTCAGCAAATCAAAGTTTAATGTCTGCTTTACTGGCGCACCCGAACCCGAGTTTGAATGCATGGGAGAGCCCAGAGTGGTG-3' (reverse primer comprising nucleotide sequence encoding a peptide F2A);

3. synthesis of a cDNA fragment of the LIN28 gene - position in the mRNA 100-796- (Homo sapiens lin-28 homolog A (C. elegans)) (registration number in GeneBank NM_024674.4) with a size of 775 nucleotides containing the sequence at the 5'-end F2A peptide (F2A-KLF4). Synthesis was carried out by polymerase chain reaction with primers: hLIN28 F2A5 '5'- GCAGACATTAAACTTTGATTTGCTGAAACTTGCAGGTGATGTAGAGTCAAATCCAGGTCCAGCTCAGCCGACGACCATGGGCTCC-3' (forward primer comprising a nucleotide sequence encoding a peptide F2A) and hLIN28 3'MluI 5'- CACTTTCTCCAACGCGTCTGCTCCTCAAAACTTCCTG-3 '(forward primer comprises artificially introduced restriction endonuclease site Mlu I);

4. The combination of the OCT4-F2A and F2A-LIN28 fragments is carried out by polymerase chain reaction using the overlapping fragments OCT4-F2A and F2A-LIN28 as well as the hOCT45'NheI and hLIN28 3'MluI primers as matrices. The result is a fragment of OCT4-F2A-LIN28, the size of 1891 nucleotides;

5. The DNA fragment encoding the DsRed2 protein is excised from the plasmid pDsRed2 (Clontech) with the Xba I restriction endonuclease, cloned into the Xba I site located in the polylinker B of the pIRES plasmid (Clontech). The result is the plasmid pIRES-DsRed2;

6. The OCT4-F2A-LIN28 DNA fragment is cloned using the pGEM-T Easy Vector Systems (Promega) reagent kit and E. coli XL-10 Gold strain;

7. clones of the pGEM-T Easy plasmid with insertions - pGEM-OCT4-F2A-LIN28 sequences are isolated by the standard alkaline lysis method (Maniatis, T., Fritsch, EF and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press);

8. the OCT4-F2A-LIN28 fragment is excised from the plasmid pGEM-OCT4-F2A-LIN28 using the EcoR I restriction endonuclease and ligated to the pIRES-DsRed2 plasmid with the hydrolyzed EcoR I restriction endonuclease, the site of which is located in polylinker A.

As a result, the plasmid pOL-DsRed2 of 8714 bp was obtained. (OL = OCT4, LIN28).

Figure 1 shows a map of the plasmid genetic construct pOL-DsRed2. The DNA fragment encoding the OCT4 and LIN28 proteins is inserted into the EcoR I site in the polylinker A of the pIRES plasmid, and the DNA encoding DsRed2 is inserted into the Xba I site of the polylinker B of the pIRES plasmid.

The description and positions in the nucleotide sequence (bp, a pair of nucleotides) of the functional elements of the genetic plasmid construct pOL-DsRed2 are presented in table 1.

Table 1 Element of plasmid genetic construct Positions in the sequence of construction, bp p CMV IE - cytomegalovirus enhancer / promoter 1-750 IVS - Intron 890-1022 T7 RNA polymerase promoter 1067-1085 DNA fragment OCT4-F2A-LIN28 1096-2984 IRES - internal ribosome landing site 3020-3601 DsRed2- cDNA of the DsRed2 gene 3624-4350 T3 RNA Polymerase Promoter 4396-4418 SV40 poly A - fragment containing the SV40 mRNA polyadenylation signal 4429-4651 Origin replication f1 4747-5203 p SV40 - enhancer / early promoter of the SV40 virus 5268-5686 SV40 ori - origin of SV40 virus replication 5584-5650 Neo-r - neomycin resistance gene 5731-6526 Synthetic polyadenylation signal 6591-6640 Amp-r - ampicillin resistance gene 7052-7913

The resulting plasmid genetic construct pOL-DsRed2 was constructed on the basis of the pIRES plasmid vector (Clontech), in which the cDNA fragments of the human OCT4 and LIN28 genes are connected, connected by the nucleotide sequence encoding the F2A peptide and the cDNA of the gene encoding the DsRed2 fluorescent protein.

Transcription of a single OCT4-F2A-LIN28-IRES-DsRed2 mRNA is carried out from the constitutive promoter p CMV IE, which provides a high level of mRNA production.

The presence of F2A and IRES sequences allows simultaneous translation of OCT4, LIN28, and DsRed2 proteins from one mRNA molecule.

The DNA fragment encoding the DsRed2 fluorescent protein is a marker for cell transfection and subsequent elimination of plasmid DNA.

The plasmid genetic construct pOL-DsRed2 is designed for temporary or permanent expression of the OCT4, LIN28 and DsRed2 genes in cultured human cells, and provides stable expression of the introduced gene.

The recombinant plasmid pOL-DsRed2 can be used as a vector to obtain induced human pluripotent stem cells.

List of references

1. Takahashi K. and S. Yamanaka. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006.126 (4): p.663-76.

2. Takahashi K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131 (5): p. 861-72.

3. Yu J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318 (5858): p. 1917-20.

4. Okita K. et al. Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008. 322 (5903): p. 949-53.

5. Szymczak A.L. and Vignali D.A. Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin Biol Ther, 2005.5 (5): p. 627-38.

6. Cowan C.A. et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med, 2004.350 (13): p. 1353-6.

Claims (3)

1. Recombinant plasmid pOL-DsRed2 encoding the human OCT4 and LIN28 proteins and the fluorescent protein DsRed2, designed to produce induced human pluripotent stem cells, which is a genetic construct based on the pIRES plasmid containing the following structural elements: DNA fragment encoding the OCT4 and LIN proteins human, including the sequence encoding F2A located between the sequences OCT4 and LIN28, is inserted into the restriction site EcoR I in the polylinker A of the pIRES plasmid, insert size 1888 nucleotides s, and a DNA fragment encoding a DsRed2 fluorescent protein inserted into the XLa I site of polylinker B of the pIRES plasmid; the transcription of polycistronic mRNA OCT4-F2A-LIN28-IRES-DsRed2 is carried out with the constitutive promoter p CMV IE. 2. The recombinant plasmid pOL-DsRed2 according to claim 1, where the separation of the nucleotide sequences by F2A and IRES elements allows the expression of three proteins from one plasmid at a time. 3. The recombinant plasmid pOL-DsRed2 according to claim 1, where the DNA fragment encoding the human OCT4 and LIN28 proteins triggers cell reprogramming to produce induced human pluripotent stem cells, and the DNA fragment encoding the DsRed2 fluorescent protein is a marker of cell transfection and subsequent elimination plasmid DNA.

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