Method for constructing fixed-point integrated exogenous DNA transgenic pigs

Abstract

The invention discloses a method for constructing fixed-point integrated exogenous DNA transgenic pigs. The method comprises the following steps: S1, performing safety target screening and target binding gRNA cleavage efficiency verification; S2, constructing a homologous arm donor plasmid and obtaining a fixed-point integrated transgenic cell line; S3, constructing the fixed-point integrated exogenous DNA transgenic pigs. The method disclosed by the invention has the benefits that a gRNA target sequence is introduced in the donor plasmid, the donor plasmid is linearized while a Cas9 nucleasecleavage target gene is induced by utilizing intracellular transcription gRNA, the test steps are significantly simplified, the labor is saved, and the co-transfection efficiency is favorably improved; carriers used for the fixed-point integration are less, a homologous arm is moderate in size, and the transgenic cell line is more favorably obtained; an efficient site-directed integration technology, a high-activity site-specific transgenic cell culture technology and a somatic cell cloning technology are combined, so that the efficient preparation of fixed-point integrated transgenic animalsis facilitated, and the breeding speed of new transgenic animal varieties is accelerated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to a method for constructing transgenic pigs with fixed-point integration of exogenous DNA, which is applied to the preparation of transgenic animals with fixed-point integration of exogenous DNA fragments, and is suitable for breeding new varieties of long-segment multi-gene fixed-point integration transgenic animals. Background technique [0002] The acquisition of large transgenic animals relies on somatic cell cloning technology (SCNT), and the successful construction of related transgenic cell lines is a key step in obtaining large transgenic animals. Traditional transgenic techniques, such as microinjection, transposon, virus vector encapsulation and infection, etc., insert the target gene into the genome in a random way. These random integrations bring many disadvantages to the establishment and breeding of transgenic animal strains in the later stage. Therefore, there is an...

AI Technical Summary

Problems solved by technology

Somatic cell cloning technology is often used for transgenic large animals such as pigs, and the cloning efficiency is only 0.5% to 1.5%. If 2H2OP technology is used, the efficiency of obtaining KI animals is only 0.03% to 0.09%, and it is difficult to obtain KI animals.

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