MAR介导的逆转录病毒包装细胞系的建立

Construction of MAR-mediated Retroviral Packaging Cell Line

根据GenBank登录的序列号,使用primer5.0进行引物设计,提取人的外周血基因组DNA为模板,PCR方法扩增出人β-珠蛋白MAR序列,将MAR序列插入到逆转录病毒表达载体pLXSN,构建MAR序列介导的逆转录病毒载体。酶切和测序鉴定后,阳离子聚合物转染PA317细胞,G418筛选出阳性细胞克隆,提取病毒液上清,测定逆转录病毒颗粒滴度,逆转录病毒颗粒的最高滴度为1.6×106CFU/mL,成功建立了MAR介导的逆转录病毒包装细胞系。

The β-globin MAR was amplified through PCR using the primer5.0 designed according to the GenBank sequence.The human genomic DNA was extracted from human blood.The cloned β-globin MAR fragment was inserted into pLXSN vector to construct the MAR-mediated retroviral vectors.After identification by restriction enzyme digestion and DNA sequencing,the recombinants were transfected into PA317 cells through positive ion liposome method.Positive cells were screened by G418,and viral titers in supernatants was harvested from culture media of G418 resistant transfected cell.Result showed that the highest viral titers is 1.6×106CFU/mL,and packaging cell line was established.