关键词: CRISPR/Cas9, 定点突变, Kcnh6基因, 糖耐量异常

Abstract: Objective To establish the Kcnh6 gene P235L site-specific mutant mice model by using CRISPR/Cas9 technique and to verify whether the phenotype is consistent with the diabetes pedigree that we found previously,in order to provide a more precise experimental tool for studying the crucial role of Kcnh6 gene in the pathogenesis of diabetes. Methods sgRNA specific for the point-mutant site was designed according to the exon 5 of Kcnh6 gene. Then,we constructed the donor vector containing the homologous arm and mutant site sequence based on the sequence of the sgRNA. Finally,sgRNA,donor vector and Cas9 were mixed together and microinjected into fertilized eggs of mice.The survived oosperms were transplanted into the uteruses of pseudopregnant mice.We detected the Kcnh6 gene mutation of the progeny by PCR and gene sequencing technology. Results We established the homozygous Kcnh6 gene P235L site-specific mutant mice. Preliminary studies showed that Kcnh6 gene knock-in site-specific mutant mice have less body weight,impaired glucose tolerance and decreased insulin secretion by feeding high fat diet,compared to wild-type mice. Conclusion We successfully constructed the homozygous Kcnh6 gene P235L site-specific mutant mice model which will provide a more precise research tool to exploit the crucial role of Kcnh6 gene in the pathogenesis of diabetes.

Key words: CRISPR/Cas9, site-specific mutation, Kcnh6 gene, impaired glucose tolerance