应用CRISPR/Cas9技术构建跨膜蛋白超家族6成员2 E167K基因敲入小鼠模型

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

目的:构建跨膜蛋白超家族6成员2(Tm6sf2) E167K基因敲入小鼠模型。方法:构建同时表达针对小鼠Tm6sf2基因特定位点的单链向导RNA Cas9的质粒和携带Tm6sf2 E167K片段的Donor质粒,将上述2质粒一起注射入小鼠受精卵,通过PCR检测和测序验证得到F0代阳性小鼠。统计F2代中野生(Wt)、杂合和敲入(KI)3种基因型小鼠的存活数量。选取F2代同窝Wt和KI雄性小鼠(8只/组)给予普通饮食8周,每周记录小鼠的体质量,检测两种小鼠葡萄糖代谢和脂质代谢等指标。组间比较采用独立样本 t检验。 结果:基因型检测和测序结果表明Tm6sf2 E167K基因敲入小鼠模型建立成功。KI小鼠不存在胚胎纯合致死的表型。哺乳期内KI小鼠较Wt小鼠的体质量升高,两组差异有统计学意义( P < 0.05)。KI小鼠的空腹血糖(9.50±0.33)mmol/L较Wt小鼠的空腹血糖(7.80±0.30)mmol/L升高,两组差异有统计学意义( P < 0.05);KI小鼠的口服葡萄糖耐量试验2 h血糖(9.20±0.51)mmol/L较Wt小鼠的口服葡萄糖耐量试验2 h血糖(7.60±0.18)mmol/L升高,两组差异有统计学意义( P < 0.05)。KI小鼠的肝脏甘油三酯含量(8.40±0.55)mg/g较Wt小鼠的肝脏甘油三酯含量(7.30±0.63)mg/g升高,但差异无统计学意义( P < 0.05);两种小鼠的血浆甘油三酯水平差异无统计学意义( P > 0.05);肝脏油红O染色结果显示KI小鼠较Wt小鼠的肝小叶中央区有更多的脂质累积。 结论:Tm6sf2 E167K基因敲入小鼠构建成功。Tm6sf2 E167K基因敲入可引起小鼠葡萄糖代谢的异常,促进肝脏脂肪变性的发生。

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.