小鼠过氧化物酶体增殖体激活受体γ_2重组逆转录病毒载体在NIH3T3细胞中的表达

Expression of mouse peroxisome proliferator activated receptor γ_2 in NIH3T3 cells induced by recombinant retrovirus vector

目的 通过应用重组逆转录病毒介导小鼠过氧化物酶体增殖体激活受体γ2(mPPARγ2 )基因整合入NIH3T3细胞基因组中并进行表达。方法 从经测序证实含正确序列mPPARγ2的重组质粒pcDNA3/mPPARγ2 中 ,双酶切下约 1.5Kb的mPPARγ2 全长cDNA编码序列 ,亚克隆入逆转录病毒载体pGCEN中构建重组逆转录病毒载体pGCEN/mPPARγ2 。pGCEN/mPPARγ2 及pGCEN经LipofectAMINE感染病毒包装细胞系PA317细胞 ,通过筛选PA317细胞G418抗性克隆 ,收集病毒上清 ,然后用其感染靶细胞NIH3T3细胞 ,用免疫荧光染色及Western印迹方法鉴定mPPARγ2 在NIH3T3细胞中的表达情况。结果 构建了含mPPARγ2 全长cDNA重组逆转录病毒载体 ,获得了滴度分别为 5×10 4 CFU/ml和 6× 10 5CFU/ml的pGCEN/mPPARγ2 及pGCEN的病毒上清。经鉴定pGCEN/mPPARγ2 能有效地感染靶细胞NIH3T3细胞并表达mPPARγ2 。结论 本研究结果为在体外建立脂肪细胞分化模型及为进一步研究PPARγ2

Objective To express the mouse peroxisome proliferator activated receptor γ 2 (mPPARγ 2) in NIH3T3 cells induced by the recombinant retrovirus vector. Methods mPPARγ 2 gene digested from the recombinant plasmid pcDNA3/mPPARγ 2 containing the target gene segment was subcloned into retrovirus vector pGCEN to generate the recombinant retrovirus pGCEN/mPPARγ 2. The recombinant retrovirus pGCEN/mPPARγ 2 and pGCEN were used to transfect PA317 cells with LipofectAMINE, and anti G418 clones of PA317 cells were selected and viral supernatants were harvested and used to infect NIH3T3 cells. The expressing products were identified with immune flurescence assay (IFA) and Western Blot. Results The recombinant retrovirus pGCEN/mPPARγ 2 was constructed, and 5×10 4 CFU/ml and 6×10 5 CFU/ml of pGCEN/mPPARγ 2 containing and pGCEN containing viral supernatants were obtained respectively. mPPARγ 2 was expressed in NIH3T3 cells mediated by the recombinant retrovirus vector. Conclusion This work is the basis for the foundation of adipocyte differentiation model in vitro and further researching on the molecular mechanism of adipocyte differentiation induced by PPARγ 2.