反转录病毒载体介导的EGFP基因在SK-N-SH神经母细胞瘤细胞中的表达

目的构建携带报告基因增强型绿色荧光蛋白(EGFP)的反转录病毒载体,并且探讨病毒载体对SK-N-SH神经母细胞瘤细胞株的感染效率。方法使用基因重组技术构建携带EGFP基因的反转录病毒载体(RV/EGFP)。将稀释的RV病毒液感染NIH3T3细胞,计数NIH3T3荧光表达细胞的数量来确定病毒的浓度;使用RV载体感染SK-N-SH细胞,通过流式细胞学荧光检测明确RV在SK-N-SH细胞的感染效率。结果成功构建了基因转移载体RV/EGFP,确定重组病毒的浓度为8.3×106病毒颗粒/mL。在SK-N-SH细胞RV/EGFP稳定转染并有效表达。在SK-N-SH细胞EGFP的荧光表达显示RV的转移并且表达的效率达到30%以上。结论SK-N-SH细胞能被RV病毒载体有效感染,在转基因细胞株外源基因的表达长期稳定并且显示RV病毒载体具有良好的基因转移效率。

Retroviral vector mediated expression of EGFP gene in SK-N-SH neuroblastoma cells

Objective To construct the retroviral(RV) vector with report gene enhanced green fluorescent protein(EGFP) and to explore the gene transfection efficiency of RV on SK-N-SH neuroblastoma cells.Methods By the technique of genetic recombination,the recombinant RV/EGFP viruses had been constructed.The concentration of diluted RV virus solution was determined by counting the NIH3T3 cells with fluorescent expression.Then the RV/EGFP virus was used to transfer SK-N-SH neuroblastoma cells.The transfection efficiency in SK-N-SH cell was measured by flow cytometer.Results The gene transfer vector RV/EGFP had been successfully constructed.We obtained the recombinant RV/EGFP with a concentration of 8.3×106 viruses/mL.The RV/EGFP had been stably transferred and effectively expressed in SK-N-SH cell.The transfection and expression efficiency of RV was above 30% in SK-N-SH cells determined by observing EGFP expression.Conclusion The SK-N-SH cells can be transferred effectively by recombinant RV vector.In transgenetic cells,the expression of transgene was stable,and moreover,RV vector had better genetically transfected efficiency than other methods.