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目的:克隆小鼠雄激素受体(androgen receptor,AR)基因的cDNA,构建AR真核表达载体,并在睾丸支持细胞TM4中进行表达和鉴定。方法:应用RT-PCR法从小鼠睾丸中扩增AR,将测序正确的PCR产物克隆至pcDNA3.1(-)真核表达载体中,再将载体以脂质体方式转染至TM4细胞系中,通过G418筛选稳定转染AR的TM4细胞株,以RT-PCR法和Western blotting法检测转染前后AR在TM4细胞系中的表达情况。结果:RT-PCR法和Western blotting法检测的结果显示,转染pcDNA3.1(-)/AR质粒的细胞中AR基因的表达水平显著高于转染pcDNA3.1(-)质粒的对照组。结论:成功构建了小鼠AR真核表达载体pcDNA3.1(-)/AR和稳定转染的TM4细胞系,为进一步研究AR在睾丸支持细胞中的作用及其分子机制建立了细胞模型。
OBJECTIVE: To clone cDNA of mouse androgen receptor (AR) gene and construct AR eukaryotic expression vector, and express and identify it in TM4 cells. Methods: AR was amplified from mouse testis by RT-PCR. The correct sequencing PCR products were cloned into pcDNA3.1 (-) eukaryotic expression vector and transfected into TM4 cell line by liposome G418 was used to screen TM4 cell line stably transfected with AR. The expression of AR in TM4 cell line was detected by RT-PCR and Western blotting. Results: The results of RT-PCR and Western blotting showed that the expression level of AR gene in pcDNA3.1 (-) / AR transfected cells was significantly higher than that in pcDNA3.1 (-) transfected cells. CONCLUSION: The mouse AR eukaryotic expression vector pcDNA3.1 (-) / AR and the stably transfected TM4 cell line were constructed successfully. Cell model was established to further study the role of AR in testicular cell support and its molecular mechanism.