ANALYSIS OF PIGGYBAC TRANSPOSON TRANSPOSITION EFFICIENCY AND INTEGRATION SITES IN BOVINE FIBROBLAST GENOME | PiggyBac转座子在牛成纤维细胞基因组中的转座效率与整合位点分析

Chinese. 内蒙古生物高新科技项目“乳腺特异性表达嗜热均糖苷酶转基因牛的研究”（20030301） 摘　　要：为分析PiggyBac转座子在牛成纤维细胞中的转座效率和整合位点。用转座子载体转染牛成纤维细胞,传代稀释比例分别为1：30、1：60、1：100,应用TailPCR技术分析PiggyBac转座子在牛基因组中的整合位点的特征。结果显示,转染转座子载体后的细胞,经筛选后到的单克隆数分别为177、134、79,当不加转座酶时得到的单克隆数分别为5、0.2、0差异极显著。在此基础上本实验进一步摸索转座子与转座酶的最佳比例,当转座子与转座酶的比率为10：1、5：1、2：1、1：5、1：10时单克隆数分别为4、89、127、131、125、86。结果显示插入位点与载体连接处都是TTAA序列,插入的位点没有处于外显子的,都是处于内含子、基因间序列或重复序列之中。40个整合位点可以定位到牛的14条染色体上。说明PiggyBac转座子能高效转座牛成纤维细胞并且能整合到基因组中。

English. In order to analysis of the PiggyBac transposon transposition efficiency and integration sites in bovine fibroblasts. Trans- fected transposon vector into the cattle fibroblasts, tile passaged dilution rates were 1：30,1：60,1：100, Application TailPCR technology analysis of the characteristics of the PiggyBac transposon integration sites in the bovine genome. The results showed that cells transfect- ed transposon vector, after screening the number of monoclonal were177,134,79, respectively, the number of monoclonal without tran- posase were 5,0.2,0, respectively, different was highly significant. On the basis of this experiment explore the optimum ratio of the transposon and transposition enzymes, when the transposon and the ratio of transposase were 10：1,5：1,2：1,1：5,1：10 the monoclonal respectively 4,89,127,131,125,86. Results showed that the insert point and vector are the TrAA sequence all of them localed in ex- ons or repeat sequences, none of the inserted were in the intron. 40 integration sites can be targeted to 14 bovine chromosomes. Piggy- Bac transposon can efficiently transpose cattle fibroblasts and can be integrated into the genome.