A construction method for site-specific integration of exogenous dna transgenic pigs

Abstract

The invention discloses a method for constructing transgenic pigs with fixed-point integration of exogenous DNA, comprising the following steps: S1, screening of safe targets and verification of target-binding gRNA cleavage efficiency; S2, constructing homologous arm donor plasmids, and obtaining fixed-point Integrating transgenic cell lines; S3, construction of exogenous DNA site-specific integration transgenic pigs. The present invention introduces the gRNA target sequence into the donor plasmid, uses intracellular transcription of the gRNA to induce Cas9 nuclease to cut the target gene, and linearizes the donor plasmid, which greatly simplifies the test steps, saves manpower, and is conducive to improving co-transfection efficiency. In the present invention, fewer vectors are used for site-specific integration, and the size of the homology arm is moderate, which is more conducive to obtaining transgenic cell lines. The combination of high-efficiency site-specific integration technology, high-activity site-specific transgenic cell cultivation technology and somatic cell cloning technology is conducive to site-specific integration of transgenic animals. Preparation to speed up the breeding of new varieties of transgenic animals.

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.