应用绿色荧光蛋白研究外源基因在造血细胞的表达调控

Employing Green Fluorescent Protein to Investigate Regulation of Exogenous Genes Expression in Hematopoietic Cells

以增强型绿色荧光蛋白为报道分子,研究双基因逆转录病毒载体介导小鼠骨髓细胞的基因转移中,SV(simianvirus)40启动子和内部核糖体进入位点(internalribosomeentrysite,IRES)对双基因共表达的影响。构建双基因中间序列分别为SV40启动子和IRES的载体pLESN和pLEIN。经包装获得较高滴度的病毒上清,以共培养的方式感染5-氟尿嘧啶预刺激的小鼠骨髓细胞。流式细胞仪检测表明转染效率约25%,PCR证明EGFP基因整合至骨髓细胞基因组。半固体培养转基因细胞7d,LEIN组获得具有G418抗性的集落中98%表达绿色荧光,而LESN组54%。结果表明:在双基因逆转录病毒载体介导的小鼠骨髓细胞的基因转导中,IRES与内部SV40启动子相比,更能保证双基因的共同表达。

It was investigated,with EGFP as a reporter,how internal promoter and IRES(internal ribosome entry site)worked after retrovirus mediated gene transfer into murine immature hematopoietic cells.Based on plasmid pLXSN and EGFP gene,pLESN and pLEIN which were distinguished in sequences between two genes,including SV40 promoter and IRES,were successfully constructed.After liposome mediated gene transfer into packaging cells,high titre virus supernatant was obtained.FACS analysis demonstrated that about 25% of murine 5 FU prestimulated bone marrow cells could be infected after cocultivation with producing cells in vitro .PCR confirmed the integration of EGFP gene into the genomic DNA of hematopoietic cells.After target cells were cultured in clonogenic progenitor assays containing G418 for 7 days,98% of G418 resistant colonies demonstrated green fluorescence in LEIN group while 54% in LESN group.Results showed that IRES was superior to SV40 promoter for guarantee of genes co expression in bicistronic retrovirus mediated gene therapy.