Gene knockout targeting vector and application thereof

A targeting vector and gene knockout technology, which can be used in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve problems such as easy loss of positive cells, and achieve the effects of convenient cryopreservation and identification, cost reduction, and high efficiency.

Inactive Publication Date: 2011-01-26
BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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  • Description
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AI Technical Summary

Problems solved by technology

At present, three research groups have successfully knocked out the PRNP gene of cattle and sheep. The vector and cell screening methods they used to enrich gene targeting efficiencies were 10.3% (55 / 533, Denning et al., 2001), 0.6%, respectively. % (1 / 163, Yu et al., 2006), 4.9% (10 / 204, Zhu, 2008), 6.4% (13 / 203, Kuroiwa, 2004), 5.2% (17 / 327, Kuroiwa, 2004), that is Said that they need to screen out a few cells with PRNP gene targeting from hundreds of cells, and it is easy to lose positive cells

Method used

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  • Gene knockout targeting vector and application thereof
  • Gene knockout targeting vector and application thereof
  • Gene knockout targeting vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of PRNP gene promoter-less targeting vector

[0029] For the construction process of the PRNP gene promoter-free targeting vector, please refer to figure 1 .

[0030] In the present embodiment, get bovine fetal skin tissue and cut into 1mm 3 The left and right small pieces were washed twice with DMEM / F12 and planted in batches in a 25cm medium containing 1mL DMEM / F12+10%FBS. 2 In the culture flask, add DMEM / F12+10% FBS to 6mL after the tissue block is firmly adhered to the wall, and store at 37°C, 5% CO 2 Cultivate in an incubator for 6-7 days, change the medium every 2 days, after the cells grow confluent, digest and passage 2-3 times with 0.25% trypsin, freeze in batches with DMEM / F12+20%FBS+10%DMSO , to obtain bovine fetal fibroblasts.

[0031] Using the genome of bovine fetal fibroblasts as a template, the 5' homology arm and the 3' homology arm were amplified, respectively.

[0032] The primer for the 5' homology arm is, LoxP-5F: 5'-TT ...

Embodiment 2

[0044] Example 2 Transfection of Bovine Fetal Fibroblasts and Cell Screening

[0045] The present invention will knock out the two alleles of the bovine PRNP gene, the liposome cell transfection method is used for the first allele knockout, and the second allele knockout is used Lonza's nuclear electrofection method.

[0046] 2.1 Knockout of the first allele of bovine PRNP

[0047] Inoculate bovine fetal fibroblasts in 6-well cell culture plates and culture them in DMEM medium containing 10% fetal bovine serum to 70-80% confluence. The linearized PRNP gene targeting vector LoxPneo-PRNP KO was transferred into cells;

[0048] After 48 hours of cell transfection, trypsinize the cells from the 6-well plate, inoculate 4×105 cells per 100 mm cell culture dish, and select the cells with G418 concentration of 600 μg / mL cell culture medium for 7-9 days;

[0049] Use a cell cloning ring to select the G418-resistant cell cloning points and inoculate them into a 24-well cell culture p...

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Abstract

The invention relates to a gene knockout targeting vector, which comprises a positive selective gene. Two ends of the positive selective gene are provided with a recombinant enzyme recognition sequence; the other end of the recombinant enzyme recognition sequence is respectively connected with a 5' homologous arm and a 3' homologous arm of a knockout gene; promoters for promoting the expression of the positive selective gene are not arranged in the two homologous arms; and the outer sides of the homologous arms are provided with negative selective gene. The targeting vector transfects cells, positive clones are screened through a culture medium containing a medicament corresponding to positive selective gene, and the obtained positive clones have high enrichment efficiency; and the cell screening method is simple without massive manpower and material resources, the subsequent cell freezing and identification are greatly facilitated, and the cost of the gene targeting is greatly reduced.

Description

technical field [0001] The invention relates to a gene knockout targeting carrier and its application, in particular to a gene knockout targeting carrier with no promoter and positive and negative screening genes and its application. Background technique [0002] After the first somatic cell gene targeting cloned sheep was born in 2000, somatic cell gene targeting combined with nuclear transfer technology has been widely used to modify the genome of large animals. Gene targeting is based on the principle of homologous recombination between DNA molecules. In somatic cells of large animals, the probability of homologous recombination is very low, which is reflected in the fact that the number of randomly integrated cells obtained through drug screening after cell transfection is far more than that of homologous recombination cells, and it is often necessary to screen a large number of cells to obtain gene targeting Cell. Although previous studies used many screening methods,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/64C12N15/85
Inventor 王少华丁方荣李松戴蕴平李宁
Owner BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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