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目的 建立并鉴定跨膜蛋白121(Tmem121)肝细胞特异性基因敲除小鼠。方法 利用CRISPR/Cas9与Cre-loxp系统构建Tmem121flox/+/Cre+与Tmem121flox/flox小鼠，再将两种小鼠进行杂交繁育获得后代小鼠。提取后代小鼠的鼠尾DNA，通过PCR鉴定小鼠基因型，获得肝细胞特异性Tmem121基因敲除小鼠(Tmem121flox/flox/Cre+,Tmem121ΔHep)。观察并分析敲除小鼠与对照小鼠的生长繁殖及器官发育情况。利用PCR及Western blot法验证Tmem121在小鼠原代肝细胞中敲除效率。利用Cell MaskTM深红质膜染色法比较对照组小鼠与敲除组小鼠原代肝细胞的形态差异并进行统计学分析。结果 通过小鼠基因型鉴定，成功获得Tmem121flox/flox/Cre+小鼠。敲除小鼠与对照小鼠在体质量、繁殖能力及肝脏的生长发育方面均无明显差异。肝细胞特异性敲除Tmem121基因对肝组织形态结构及病理特征无明显影响。与对照组相比，敲除组小鼠原代肝细胞Tmem121在mRNA及蛋白水平均明显降低(P<0.01)。Cell MaskTM深红质膜染色结果显示Tmem121敲除组小鼠的原代肝细胞的双核比例增多(P<0.05)，细胞表面积减小(P<0.001)。结论 成功建立Tmem121肝细胞特异性基因敲除小鼠模型，为进一步研究Tmem121基因在肝脏疾病中的作用及机制提供了动物模型支持。
Abstract：Objective To establish and identify hepatocyte-specific transmembrane protein 121(Tmem121)knockout mice. Methods The hepatocyte-specific Tmem121 knockout mice(Tmem121flox/flox/Cre,Tmem121ΔHep)were obtained by crossbreeding of Tmem121flox/+/Cre and Tmem121flox/floxmice,which were generated using the CRISPR/Cas9 and Cre/Loxp systems. The genotype was verified by PCR using genomic DNA extracted from mouse tails as template. The growth,reproduction and organ development of both control and knockout mice were observed and analyzed. PCR and Western blot methods were performed to assess the knockout efficiency of Tmem121in mouse primary hepatocytes. CellMaskTMDeep Red plasma membrane staining was employed to compare the morphological differences in primary hepatocytes between control and knockout mice. Results Tmem121flox/flox/Cre mice were successfully obtained according to genotype identification analysis,and there were no significant differences between control and knockout mice in body mass,reproductive ability,growth and development of liver. The specific knockout of Tmem121 gene in primary hepatocytes did not significantly affect the morphological structure or pathological characteristics of liver tissue. However,compared to the control group,the levels of Tmem121 mRNA and protein in the primary hepatocytes of the knockout group were significantly reduced(P < 0. 01). CellMaskTMDeep Red plasma membrane staining indicated that the proportion of binucleated hepatocytes in Tmem121-deficient mice significantly increased(P < 0. 05),while the cell area was significantly reduced(P < 0. 001). Conclusion Hepatocyte-specific Tmem121 knockout mice are successfully constructed,which provides an animal model for further exploration of the function and mechanism of Tmem121 gene in liver diseases.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2025.09.004
中图分类号:R-332;R575
引用信息:
[1]王月,何国良,李兰玉,等.Tmem121肝细胞特异性敲除小鼠模型的建立与鉴定[J].安徽医科大学学报,2025,60(09):1591-1598.DOI:10.19405/j.cnki.issn1000-1492.2025.09.004.
基金信息:
国家自然科学基金项目(编号：82060124、82260529); 广西科技厅中央引导地方科技发展资金项目(编号：桂科ZY21195024); 大学生创新创业训练计划项目(编号：202210601005)~~