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Plasmid vector and application thereof in site-specific integration of exogenous genes at targeted pig COL1A1 locus

A plasmid vector, foreign gene technology, applied in the field of genetic engineering, can solve problems such as difficult to achieve

Pending Publication Date: 2021-05-28
成都中科奥格生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Rosa26 is a friendly gene expressed systemically and has its own Rosa26 promoter. The gene inserted at this site often leads to widespread expression of the gene throughout the body. However, in practice, transgenic technology sometimes requires tissue-specific expression. , this site is difficult to achieve

Method used

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  • Plasmid vector and application thereof in site-specific integration of exogenous genes at targeted pig COL1A1 locus
  • Plasmid vector and application thereof in site-specific integration of exogenous genes at targeted pig COL1A1 locus
  • Plasmid vector and application thereof in site-specific integration of exogenous genes at targeted pig COL1A1 locus

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Experimental program
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Effect test

Embodiment 1

[0044] Preparation of transgenic pigs with site-specific integration of human thrombomodulin TM at COL1A1 site

[0045] 1. Design the sgRNA that specifically recognizes the target sequence at the COL1A1 site of the pig genome, and connect it into the pX458 vector recovered after digestion with Bbs I to construct a knockout vector for the COL1A1 target site (such as image 3 shown).

[0046] Obtain exogenous gene fragments by means of Overlap PCR and enzyme-cut ligation, introduce restriction sites EcoRI and NoT I at both ends, and construct a site-directed integration vector containing COL1A1 sites with homology arms on both sides (the diagram is as follows: Figure 4 ).

[0047] (1) Construction of pX458-Col1a1 knockout vector:

[0048] a. The sgRNA oligonucleotide chain anneals to form a double-stranded fragment with sticky ends

[0049] Table 1 Oligonucleotide sequence information

[0050]

[0051] Take the forward and reverse oligonucleotide chains of sg-Col1a1-targ...

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Abstract

The invention relates to the technical field of gene engineering, and particularly relates to a plasmid vector and application thereof in site-specific integration of exogenous genes in a downstream sequence (hereinafter referred to as COL1A1 locus) of a targeted pig COL1A1 gene. The plasmid vector provided by the invention comprises a COL1A1 locus homologous left arm, an exogenous gene and a COL1A1 locus homologous right arm. The vector is designed based on a CRISPR / Cas9 targeting system, and the vector can accurately introduce the exogenous genes into the COL1A1 locus of a pig so as to solve the problems that the traditional transgenic technology is low in efficiency, the exogenous genes are randomly integrated, the difference of expression quantities of different individuals is great, and the integration position detection difficulty is large.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a plasmid vector and its application in targeting porcine COL1A1 site for site-specific integration of exogenous genes. Background technique [0002] Transgenic pigs play an important role in agricultural and medical research. On the one hand, pigs are an important species for animal husbandry. On the other hand, pigs and humans are closer to other animals such as humans and mice, and they also play a significant role in medical research. For example, the size of organs between pigs and humans Close, this makes xenotransplantation possible, and the structure of various organs of pigs and humans is similar to the physiological and biochemical indicators of blood, and transgenic pigs become a better animal model. [0003] Transgenic pigs have been applied in many fields, but they are still subject to certain limitations. The main reasons are the uncertainty of transgen...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N15/90A01K67/027
CPCC12N15/8509C12N15/113C12N15/907A01K67/0276A01K67/0278C12N2310/20A01K2207/15A01K2217/072A01K2217/075A01K2227/108A01K2267/02
Inventor 潘登科杜嘉祥王永
Owner 成都中科奥格生物科技有限公司
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