Method for constructing Fzd6-Q152E site-directed mutagenesis mouse model based on CRISPR/Cas9 and application

Abstract

The invention discloses a method for constructing an Fzd6-Q152E site-directed mutagenesis mouse model based on CRISPR/Cas9 and application. It is found through comparison that the positions of rs61753730 in different species have certain conservative property. Therefore, the method comprises the steps of targeting positions of rs61753730, namely Q152E, in a mouse, designing andn synthesizing sgRNA, corresponding primers and Donor sequences, constructing an sgRNA/Cas9 expression vector, then performing in-vitro transcription after the vector is subject to linearization and purification, then mixing sgRNA, Cas9mRNA and Donor, putting the mixture into fertilized eggs of the mouse is through micro-injection, and then transplanting the survived fertilized eggs into the uterus of a pseudopregnant female mouse, wherein the born mouse is an edited F0-generation mouse; and mating a positive mouse with a wild mouse to obtain F1-generation mice through PCR and Sanger sequencing identification, finally mating F1-generation heterozygous mice which are identified to be positive, and breeding the obtained progeny to acquire a homozygous Fzd6-Q152E mutant and wild mouse. The model mouse can be used for researching the biological function of rs61753730.

Description

technical field [0001] The invention belongs to the field of biotechnology research, and in particular relates to a method and application for constructing a Fzd6-Q152E site-directed mutation mouse model based on CRISPR / Cas9. Background technique [0002] Single nucleotide polymorphism (Single nucleotide polymorphism, SNP) is a third-generation genetic marker, mainly referring to the polymorphism of DNA sequence caused by a single nucleotide variation at the genomic level, that is, a specific nucleotide in the genome There are two different nucleotide changes at the acid position, and some changes can cause differences in the encoded amino acids. SNPs are widespread in the human genome, with an average of one in every 500-1000 base pairs, and this nucleotide variation is the most common type of human heritable variation, which has a role in the genetics of diseases Significance, therefore, it is very important to deeply mine SNPs and study their biological functions. [00...

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At present, some studies have found that rs61753730 may be involved in the formation of huma...

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