转MSTN干扰载体细胞株的获得及外源基因整合情况的研究

转基因细胞株的建立能够为转基因体细胞克隆技术奠定重要基础。该实验利用MSTN干扰载体,转染鲁西黄牛胎儿成纤维细胞,获得5个相应的转基因单克隆细胞株。采用Realtime PCR和高效热不对称互交式PCR（hiTAIL-PCR）技术检测细胞克隆中MSTN表达载体的拷贝数及其在牛基因组中的整合位点。结果表明,荧光定量PCR有效检测到5个细胞克隆中质粒的拷贝数分别为2.26±0.32、1.52±0.25、25.68±1.02、8.43±0.73和6.72±0.10。hiTAIL-PCR对整合位点的检测结果表明,质粒片段在插入到基因组的过程中进行了重组,其与基因组的结点处有2或4个共同的碱基序列。该研究探索MSTN干扰载体在牛胎儿成纤维细胞中的整合机制,以期获得遗传背景清楚的转基因细胞作为体细胞核移植的重要材料,为高产转基因肉牛新品种的培育提供重要的理论和实验基础。

Establishment of MSTN Interfering Plasmid Transgenic Cell Lines and Integration of Exogenous Gene

In this study, we used MSTN interference vector to transfect Luxi cattle fetal fibroblasts and obtained five transgenic monoclonal cell lines. By Real-time PCR and high-efficiency thermal asymmetric interlaced PCR, we detected the expressing copies of MSTN plasmid and their integration sites in bovine genome. The results showed that the copy numbers of plasmid were effectively detected by quantitative PCR in the five positive cell clones which were 2.26±0.32, 1.52±0.25, 25.68±1.02, 8.43±0.73 and 6.72±0.1, respectively. Integration sites detected by hiTAIL-PCR showed that there was recombination in the process of insertion of plasmid fragment into target genome. This study explored the integration mechanism of MSTN carrier interference in bovine fetal fibroblasts, in order to obtain the clear genetic background of transgenic cells. It is important for obtaining transfer material by somatic cell nuclear, providing important theoretical and experimental basis for cultivation of highyielding new varieties of transgenic cattle.