小鼠甲状腺转录因子-2转基因动物表达载体的构建及其在骨髓间充质干细胞中的表达

Construction of Transgenic Animal Expression Vector of Mouse Thyroid Transcription Factor-2 and Its Expression in Bone Marrow-derived Mesenchymal Stem Cells

目的构建小鼠甲状腺转录因子-2(TTF-2)转基因动物表达载体(pBROAD3-TTF-2),观察其在小鼠骨髓间充质干细胞(BMSC)中的表达。方法从C57BL/6J小鼠肝脏组织中提取基因组DNA,利用聚合酶链式反应方法扩增出TTF-2基因1 113 bp开放阅读框,通过DNA重组技术将TTF-2基因片段插入克隆载体pMD18-T中,经测序正确后,再重组于pBROAD3-mcs中,构建转基因动物表达载体pBROAD3-TTF-2,用酶切电泳分析对其进行鉴定。运用脂质体转染试剂将其转染BMSC后,蛋白质印迹法检测TTF-2基因的表达。结果①DNA测序证实目的基因序列正确无突变,酶切电泳分析得到相应的目的片段,大小与理论计算值一致,成功构建转基因动物表达载体pBROAD3-TTF-2。②蛋白质印迹法显示转染的BMSC高表达TTF-2蛋白。结论成功构建了pBROAD3-TTF-2转基因动物表达载体,显示其转染BMSC后TTF-2基因的表达,为下一步建立TTF-2转基因小鼠模型奠定了基础。

Objective To construct the transgenic animal expression vector （pBROAD3-TTF-2） of mouse thyroid transcription factor-2 （TTF-2） and to observe its expression in bone marrow-derived mesenchymal stem cells （BMSCs）. Methods Genomie DNA was extracted from C57BL/6J mouse liver tissue, and the 1 113 bp open reading frame （ORF） of TTF-2 gene was amplified with PCR. The amplified gene was inserted into clone vector pMD18-T, which was identified by DNA sequencing. The gene was recombinated to vector pBROAD3-mcs to construct the transgenic animal expression vector pBROAD3-TTF-2, which was identified by restriction enzyme analysis. The vector pBROAD3-TTF-2 was transfected into BMSCs by LipGenTM-mediated transfer method. The expression of TTF-2 protein in transfected BMSCs was detected by using Western Blotting. Result The result of DNA sequencing analysis and electrophoresis confirmed the vector pBROAD3- TTF-2 was successfully constructed. After transfection, the vector pBROAD3-TTF-2 could highly express in BMSCs by Western Blotting. Conclusion The transgenic animal expression vector pBROAD3-TTF-2 has been successfully constructed and highly expressed in BMSCs obtained, which lays a foundation for further generation of TTF-2 transgenic mice.