Fixed-point gene editing method based on intratestis injection

Abstract

The invention discloses a simple, convenient and efficient fixed-point gene editing method relating to the fields of transgenic animal preparation, gene functional study and biomedicines. A testis injection method is a sperm-mediated gene transferring method developed by utilizing the sperm capacity of actively combining, transferring and integrating an exogenous DNA. By using the method disclosed by the invention, an animal testis injection method is optimized, a carrier constructed by utilizing a clustered regularly interspaced short palindromic repeat system (CRISPR/Cas9) is integrated with a sperm chromosome through multi-point testis injection or seminiferous tubule microinjection, so that the aims of deleting, replacing and inserting genes are achieved. Then, a transgenic animal descendant is obtained through various approaches such as natural mating and artificial insemination. According to the method, the advantages of a CRISPR/Cas9 gene editing system are sufficiently utilized, and the existing transgenic animal system is combined, so that the complexity of in-vitro cell operation and requirement on expensive precise instruments/equipment are avoided while the gene modification animal obtaining efficiency is greatly increased.

Description

technical field [0001] The invention relates to a technique for gene-directed editing through testicular injection of mice, and belongs to the field of animal genetics and breeding. Background technique [0002] Transgenic animals refer to genetically engineered animals that stably integrate exogenous genes introduced by experimental methods in the genome, and the exogenous genes can be stably passed on to offspring. Because transgenic animals have broad application prospects in developmental biology and basic genetics research, breeding new animal species, large-scale production of medicinal proteins, and production of replanted organs, etc., they have become a hot spot in current research. [0003] Sperm has the ability to actively combine, transport, and integrate foreign DNA, and it can be introduced into oocytes during fertilization to obtain transgenic animals. Sperm-mediated gene transfer (Sperm-mediated gene transfer, SMGT) is currently one of the simple and efficie...

AI Technical Summary

Problems solved by technology

[0005] At present, gene editing technology mainly injects artificially designed plasmid vectors into embryos through microinjectio