CRISPR Cas9 conditional gene knockout mouse and establishment method

Abstract

The invention belongs to the technical field of gene knockout, and discloses a CRISPR Cas9 conditional gene knockout mouse and an establishment method. According to the establishment method, a vectoris designed and constructed, and gRNA and Cas9 protein are prepared; a gRNA sequence and a targeting vector are designed; the targeting vector is constructed by the In-Fusion technology, and the targeting vector is verified by restriction digestion, PCR and sequencing; the gRNA and Cas9 protein are prepared at the same time; microinjection and F0 mouse identification are performed; the obtained Donor vector, gRNA and Cas9 protein are co-injected into a fertilized egg; the fertilized egg after microinjection is returned to the oviduct of a surrogate mouse; PCR and sequencing identification areperformed after a mouse is born, and a positive F0 mouse is obtained; the reproduction and identification of a F1 generation mouse are performed; the sexually mature positive F0 mice are matched withwild-type mice respectively for reproducing the first generation, and a polycystic kidney gene PKD1 knockout mouse is obtained successfully.

Description

technical field

[0001] The invention belongs to the technical field of gene knockout, and in particular relates to a CRISPR Cas9 conditional gene knockout mouse and a method for establishing it. Background technique

[0002] Gene knockout (knockout) is a kind of exogenous DNA introduction that uses a DNA fragment containing a certain known sequence to undergo homologous recombination with a gene with the same or similar sequence in the genome of the recipient cell, integrates into the genome of the recipient cell, and is expressed technology. It is aimed at a sequence whose sequence is known but whose function is unknown, changing the genetic gene of the organism, making the specific gene function ineffective, so that part of the function is blocked, and can further affect the organism, and then infer the gene's function biological functions. Gene knockout and gene insertion technologies are the latest exogenous DNA introduction technologies that emerged in the 1990s. Gen...

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