[Construction of transmembrane 6 superfamily member 2 E167K gene knock-in mouse model by using CRISPR/Cas9 technology].

ABSTRACT

Objective: To construct a transmembrane 6 superfamily member 2 (Tm6sf2) E167K gene knock-in mouse model. Methods: The plasmid was constructed to simultaneously express the single-stranded guide RNA Cas9 at a specific site of the mouse Tm6sf2 gene in the donor plasmid carrying the Tm6sf2 E167K fragment. The above two plasmids were injected into the mouse fertilized eggs together. The positive F0 generation mice were validated by PCR detection and sequencing. The number of F2 generation surviving mice in three genotypes of wild (Wt), heterozygous and knock-in (KI) were calculated. Wt and KI male mice (8 mice/ group) of F2 generation littermates were selected and given a normal diet for 8 weeks. The body weight of the mice was recorded every week, and the glucose metabolism and lipid metabolism indexes of the two mice were detected. The comparison between groups was performed with an independent sample t-test. Results: Genotype detection and sequencing results showed that the Tm6sf2 E167K gene knock-in mouse model was successfully established. KI mice had absence of homozygous lethal embryo phenotype. The body weight of KI mice was higher than that of Wt mice during lactation, and the difference between the two groups was statistically significant (P < 0.05).The fasting blood glucose of KI mice (9.50 ± 0.33)mmol/L was higher than that of Wt mice (7.80 ± 0.30)mmol/L, and the difference between the two groups was statistically significant (P < 0.05). During the oral glucose tolerance test, the 2-hour blood glucose level of KI mice (9.20 ± 0.51)mmol/L was higher than that of Wt mice (7.60 ± 0.18)mmol/L, and the difference between the two groups was statistically significant (P < 0.05). The liver triglyceride content of KI mice (8.40 ± 0.55)mg/g was higher than that of Wt mice (7.30 ± 0.63)mg/g, but the difference was not statistically significant (P > 0.05). There was no significant difference in plasma triglyceride levels between the two mice (P > 0.05). The Oil red O staining results showed that KI mice had more lipid accumulation in the centrilobular region of ​​liver than Wt mice. Conclusion: Tm6sf2 E167K gene knock-in mice were successfully constructed. Tm6sf2 E167K gene knock-in can cause abnormal glucose metabolism in mice and promote the occurrence of hepatic steatosis.