Construction and phenotypic verification of PCSK9 gene knockout mouse model based on CRISPR/Cas9 technology
Objective To establish a mouse model of preprotein convertase subtilisin/kexin type 9(PCSK9)gene knockout using CRISPR/Cas9 technology and to preliminarily validate its phenotype.Methods A single-stranded guide RNA(sgRNA)was designed for the target sequence of the PCSK9 gene through non-homologous end joining(NHEJ)repair and mutation.The sgRNA and Cas9 mRNA were microinject-ed into fertilized eggs of C57BL/6J mice to generate FO generation positive knockout mice,followed by breeding and genotype identification to obtain homozygous PCSK9 knockout(PCSK9 KO)mice.PCR a-nalysis was employed for genetic identification of PCSK9 KO mice,while western blotting was utilized to assess protein expression levels of PCSK9 and LDL-R in liver tissue,and the expression levels of PCSK9 protein in heart,brain,kidney and adipose tissues were detected.Additionally,q-PCR was performed to measure the mRNA expression level of PCSK9.Enzymatic methods were used for quantification of total cholesterol(TC)and triglyceride(TG)content in serum.Results The gel electrophoresis results of PCR products,along with the analysis of PCSK9 protein and mRNA expression,successfully demonstrated the generation of systemic PCSK9 knockout mice.The expression of LDL-R(3.42±0.56)protein in liver tis-sue was significantly increased.Compared with the WT group,the serum TC(90.75±17.81 mg/dL vs 54.19±2.42 mg/dL)and TG(81.26±11.98 mg/dL vs 50.92±11.93 mg/dL)of PCSK9 mice were sig-nificantly reduced(P＜0.001).Conclusion The PCSK9 knockout mice were successfully generated using CRISPR/Cas9 technology.Knocking out the PCSK9 gene results in a substantial were decreased in lipid levels by upregulating liver LDL-R protein.
万方数据
维普
