绵羊TERT基因修饰骨髓间充质干细胞生物学特性

BIOLOGICAL CHARACTERISTICS OF BONE MARROW MESENCHYMAL STEM CELLS MODIFIED BY TELOMERASE REVERSE TRANSCRIPTASE GENE IN SHEEP

目的将绵羊端粒逆转录酶(TERT)基因表达载体pcDNA3.1-EGFP-TERT转入绵羊骨髓间充质干细胞(BMSCs)中,进一步研究TERT在BMSCs永生化过程中的作用。方法用RT-PCR技术扩增出带EcoRⅠ和HindⅢ酶切位点的TERT基因,连接到pcDNA3.1-EGFP真核表达载体。脂质体转染到BMSCs中,G418筛选出转染成功细胞,进行传代培养、生长曲线的测定以及遗传稳定性分析。结果扩增得到的TERT基因cDNA全长为1 866bp,转染BMSCs以后传至第40代时,核型分析正常率为77.78%。结论成功构建了绵羊pcDNA 3.1-EGFP-TERT真核表达载体,TERT-BMSCs传至40代时仍有较高的核型正常率。

Objective The eukaryotic expression vector-pcDNA3.1-EGFP-TERT-was transferred into sheep bone marrow mesenchymal stem cells（BMSCs）to further study the role of the cells in the process of permanent biochemistry. Methods Applying RT-PCR technique,the TERT gene with EcoR Ⅰ and HindⅢ enzyme loci was amplified and connected to pcDNA3.1-EGFP eukaryotic expression vector.Liposome was then transfected into BMSCs,and successfully transfected cells were screened out from G418 cells,subculture,growth curve and genetic stability test were carried out. Results The cDNA TERT gene length was 1 866 bp.After transfection of BMSCs to 40th generation,karyotype analysis showed that the normal rate was 77.78%. Conclusion A sheep pcDNA3.1-EGFP-TERT eukaryotic expression vector has been successfully created,when TERT-BMSCs is transferred to 40th generation,it still has a high normal karyotype.