双基因导入人脐血CD34^+细胞共表达的调控

Regulation of gene co expression after bicistronic retrovirus mediated gene transfer into human cord blood CD34^+ cells

目的 观察携带双基因的逆转录病毒载体转导人脐血CD3 4+细胞后 ,猴病毒 4 0 (SV4 0 )早启动子和内部核糖体进入位点 (IRES)对载体上、下游双基因共表达的影响。方法 分别构建含SV4 0早启动子和IRES的双基因逆转录病毒载体pLESN和pLEIN ,两者均携带增强型绿色荧光蛋白和新霉素抗性neo基因。重组载体经包装以其病毒上清感染经磁性细胞分离仪富集的人脐血CD3 4+细胞 ,而后进行集落形成细胞测试。结果 流式细胞仪检测表明 10 %的脐血CD3 4+细胞表达绿色荧光蛋白。集落形成细胞测试发现pLESN转导的脐血CD3 4+细胞体外培养形成的G4 18抗性集落中 ,4 1 1%的集落有绿色荧光 ,而pLEIN组全部为绿色荧光集落。结论 与SV4 0早启动子相比 ,含有IRES序列的重组双基因逆转录病毒载体转导人脐血CD3 4+细胞时 ,能保证上、下游双基因的协同表达。

Objective To compare internal SV40 early promoter with IRES of the in regulatory effect on gene co expression after retrovirus mediated gene transfer into human cord blood CD34 + cells Methods Based on pLXSN , vectors pLESN and pLEIN carrying EGFP and neo gene, distinguished by the in sequences between two, genes, were constructed After liposome mediated gene transfer into packaging cells, high titre virus supernatant was obtained After supernatant infection in vitro, transduced cells were cultured in CFC assay Results FACS analysis demonstrated that 10% of human cord blood CD34 + cell were infected in vitro All of G418 resistant colonies demonstrated green fluorescence in LEIN group, while only 41 1% in LESN group Conclusion IRES is superior to SV40 promoter for the guarantee of co expression in bicistronic retrovirus mediated gene therapy in human cord blood CD34 + cells