肝脏GPAT4基因敲除小鼠模型构建

Construction of liver GPAT4gene knockout mouse model

目的为胰岛素抵抗及非酒精性脂肪肝(NAFLD)发病与甘油-3-磷酸酰基转移酶-4(GPAT4)后续相关研究提供肝脏GPAT4基因敲除小鼠模型。方法选择C57BL/J6雌性与雄性小鼠各10只及白蛋白启动子调控的Cre转基因(Alb-Cre)小鼠作为研究对象。采用Cre/LoxP基因打靶策略,构建基因打靶载体,引入LoxP位点,获得基因同源重组胚胎干细胞(ES);将ES植入小鼠,分别获得嵌合鼠、杂合子、纯合子小鼠。最终获得肝脏特异性GPAT4敲除小鼠。基因分型实验对第8外显子下游的loxP位点设计基因型鉴定引物,以便进行批量快速基因型分析;随机抽取3只小鼠进行解剖,从高表达GPAT4的小鼠肝脏、白色脂肪和棕色脂肪提取总RNA,通过实时荧光定量聚合酶链式反应(qRT-PCR)检测组织中GPAT4mRNA表达;在小鼠的肝脏组织中提取总蛋白,使用蛋白质印迹法检测肝脏GPAT4表达,进一步验证肝脏GPAT4的敲除效果。结果成功组装靶向载体,且GPAT4基因位于小鼠第8号染色体;酶切分析结果显示,6条带为线性后的靶标载体,线性载体在电泳中相对分子质量最大、速度最慢;导入ES后,确认了6个潜在的靶向克隆,即2A2、2A4、2C7、2C8、2F8和2G11。基因分析结果显示,编号3、6小鼠为肝脏特异性GPAT4敲除小鼠。qRT-PCR检测结果显示,肝脏组织GPAT4特异敲除雄鼠与雌鼠的GPAT4mRNA表达量分别为0.88±0.24和0.49±0.13,低于小鼠GPAT4被loxP两侧标记(GPAT4-FLOX)的11.24±1.43和10.78±1.98,差异有统计学意义,t值分别为-22.594和-16.399,均P<0.001;而棕色脂肪及白色脂肪组织中的GPAT4mRNA的表达均没有受到影响,差异无统计学意义,均P>0.05。蛋白质印迹法检测结果显示,GPAT4-FLOX小鼠肝脏细胞内GPAT4的表达正常,而GPAT4肝脏特异性敲除小鼠的肝脏细胞内GPAT4不表达。结论成功建立了肝脏特异性GPAT4敲除小鼠模型,为后期相关研究提供可靠的动物模型。

Objective To provide a liver glycerol-3-phosphate acyltransferase-4(GPAT4)gene knockout mouse model for the pathogenesis of insulin resistance and non-alcoholic fatty liver disease(NAFLD),as well as subsequent studies on GPAT4.Methods Ten C57BL/J6female and ten male mice,as well as albumin promoter regulated Cre transgenic(Alb-Cre)mice were selected as the research subjects.Using the Cre/loxP gene targeting strategy,agene targeting vector was constructed,and the LoxP site was introduced to obtain homologous recombinant embryonic stem cell(ES).We implanted ES cells into mice to obtain chimeric,heterozygous,and homozygous mice,respectively.Finally,liver specific GPAT4 knockout mice were obtained.Genotyping experiments were conducted to design genotype identification primers for the loxP site downstream of exon 8,in order to conduct rapid genotype analysis in batches.Three mice were randomly selected for dissection,and total RNA was extracted from the liver,white fat,and brown fat of mice with high expression of GPAT4.GPAT4mRNA expression in tissues was detected by quantitative real-time polymerase chain reaction(qRTPCR).Western blotting was used to extract total protein from mouse liver tissue and detect liver GPAT4expression to further validate the knockout effect of liver GPAT4.Results The targeted vector was successfully assembled,and the GPAT4gene was located on chromosome 8of the mouse.The results of enzyme digestion analysis showed that the six bands were linear target carriers,and the linear carriers had the highest relative molecular weight and slowest speed in electrophoresis.After importing ES cells,six potential targeted clones were identified,namely 2A2,2A4,2C7,2C8,2F8,and 2G11.The genetic analysis results showed that mice numbered 3and 6were liver specific GPAT4knockout mice.The qRT-PCR detection results showed that the GPAT4mRNA expression levels in liver tissue of GPAT4specific knockout male and female mice were 0.88±0.24and 0.49±0.13,respectively,lower than 11.24±1.43and 10.78±1.98in GPAT4-flanked by loxP(GPAT4-FLOX)mice,with statistically significant differences.The t-values were-22.594and-16.399,respectively,with P<0.001.The expression of GPAT4mRNA in brown adipose tissue and white adipose tissue was not affected,and the difference was not statistically significant,both P>0.05.The results of protein blotting showed that GPAT4expression was normal in the liver cells of GPAT4-FLOX mice,while GPAT4was not expressed in the liver cells of GPAT4liver specific knockout mice.Conclusion The results of this experiment prove that we have successfully established a liver specific GPAT4knockout mouse model,which will provide a reliable animal model for further related research.