一种真核生物染色质隔离子筛选方案的初步建立

目的 探讨建立染色质隔离子的正负筛选工具的可行性.方法 合成引物从鸡基因组cHS4区域扩增了1段特异的DNA,将PCR产物克隆到T-载体后测序,验证其对增强子的阻断功能;对pEGFP-N1载体进行一系列改造,评价其正负筛选效果. 结果 成功地获得已知的隔离子片段,初步结果表明该片断能阻断增强子的转录激活作用.将TK基因作为负向筛选标记引入pEGFP-N1载体后获得了改造后的载体,转染COS-7细胞经过G418筛选10d后获得了稳定转染的细胞系,在不同浓度GCV药物作用下细胞杀伤效果有差异. 结论 正负筛选方法被成功地引入到筛选系统,为进一步进行染色质隔离子的筛选与利用奠定了基础.

Construction of screen system for chromosome insulator in eukaryotes

Objective To further investigate the function of chromatin insulators and to construct a screen system for chromatin insulators. Methods A primer set targeting chicken cHS4 locus was designed and synthesized. The specific PCR product using this primer set was obtained and sequenced. In order to test its blocking activity, we further inserted this fragment between enhancer and promotor. Then TK gene was inserted into modified pEGFP-N1 plasmid, which was tested for its negative and positive selection. Results The result of luciferase activity showed that insulators could function when it was located on plasmid. Then vector pEGFP-N1 was modified as a screening system for chromatin insulator, in which phosphotransferase gene and thymidine kinase gene could function as selection marker. In order to evaluate its efficiency and viability, the recombinant vector was transfected into COS-7 cells. After selected with G418 for 10 d, stable transfected cells with G418 resistance were obtained. The sensitivity to pro-drug of GCV of these cells was different from that of its control cells by MTT. Conclusion This study provides an early research to further screen and utilize chromatin insulator in eukaryotes.