Construction of liver GPAT4 gene knockout mouse model
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/J6 female 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,a gene 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 select-ed for dissection,and total RNA was extracted from the liver,white fat,and brown fat of mice with high expression of GPAT4.GPAT4 mRNA expression in tissues was detected by quantitative real-time polymerase chain reaction(qRT-PCR).Western blotting was used to extract total protein from mouse liver tissue and detect liver GPAT4 expression to further validate the knockout effect of liver GPAT4.Results The targeted vector was successfully assembled,and the GPAT4gene was located on chromosome 8 of 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 e-lectrophoresis.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 3 and 6 were liver specific GPAT4 knockout mice.The qRT-PCR detection results showed that the GPAT4 mRNA expression levels in liver tissue of GPAT4 specific knock-out male and female mice were 0.88±0.24 and 0.49±0.13,respectively,lower than 11.24±1.43 and 10.78±1.98 in GPAT4-flanked by loxP(GPAT4-FLOX)mice,with statistically significant differences.The t-values were-22.594 and-16.399,respectively,with P＜0.001.The expression of GPAT4 mRNA in brown adipose tissue and white adipose tis-sue was not affected,and the difference was not statistically significant,both P＞0.05.The results of protein blotting showed that GPAT4 expression was normal in the liver cells of GPAT4-FLOX mice,while GPAT4 was not expressed in the liver cells of GPAT4 liver specific knockout mice.Conclusion The results of this experiment prove that we have suc-cessfully established a liver specific GPAT4 knockout mouse model,which will provide a reliable animal model for further related research.
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