Retrovirus-mediated gene transfer to cystic fibrosis airway epithelial cells: effect of selectable marker sequences on long-term expression.
Retrovirus-mediated gene transfer to cystic fibrosis airway epithelial cells: effect of selectable marker sequences on long-term expression.
复制标题
逆转录病毒介导的基因转移至囊性纤维化气道上皮细胞：选择标记序列对长期表达的影响。
DOI：
10.1093/nar/21.3.663
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发表时间：
1993
影响因子：
14.9
通讯作者：
Boucher,RC
中科院分区：
文献类型：
--
作者：
Olsen,JC;Johnson,LG;Wong-Sun,ML;Moore,KL;Swanstrom,R;Boucher,RC
逆转录病毒介导的基因转移提供了在载体DNA整合到宿主基因组中之后转导基因在宿主细胞中稳定长期表达的潜力。使用鼠嗜中性逆转录病毒载体，含有白细胞介素-2受体（IL-2 R）基因作为报告基因和新霉素磷酸转移酶（neorr）基因作为显性选择标记，我们测量了逆转录病毒介导的基因转移的效率和转导的基因在囊性纤维化气管上皮细胞系（CFT 1）中表达的稳定性。使用IL-2 R细胞表面标记物作为感染的报告者允许定量载体基因表达和用载体转导的细胞的流式细胞术分选。在最初的研究中，确定了逆转录病毒介导的基因转移的最佳条件。最佳转导效率需要聚阳离子的存在。通过重复暴露于病毒，CFT 1细胞的感染效率增加，使得在连续三天暴露后，可以使约80%的细胞死亡。还评价了非选择性IL-2 R基因表达的长期稳定性。表达IL-2 R的细胞百分比缓慢下降，这些细胞保持在恒定的选择压力下表达新基因，该基因由内部启动子表达。当最初选择培养物进行新基因表达，然后不进行选择而继续培养时，也得到了类似的结果。相反，在不存在G418选择的情况下，在多次感染后至少一年内在CFT 1细胞中观察到稳定表达。总之，（i）在适当的聚阳离子和多次暴露的存在下，使用嗜酸性逆转录病毒载体将外源基因转导到人气道上皮细胞中可以是高效的;和（ii）在没有选择的情况下，在这些上皮细胞中更好地维持非选择基因的稳定表达。
Retrovirus-mediated gene transfer offers the potential for stable long-term expression of transduced genes in host cells subsequent to integration of vector DNA into the host genome. Using a murine amphotropic retrovirus vector containing an interleukin-2 receptor (IL-2R) gene as a reporter and a neomycin phosphotransferase (neorr) gene as a dominant selectable marker, we measured the efficiency of retrovirus-mediated gene transfer and the stability of transduced gene expression in a cystic fibrosis tracheal epithelial cell line (CFT1). The use of the IL-2R cell surface marker as a reporter of infection permitted both quantitation of vector gene expression and flow cytometric sorting of cells transduced with the vector. In initial studies, the optimal conditions for retrovirus-mediated gene transfer were determined. The presence of a polycation was required for optimal transduction efficiency. The efficiency of infection of CFT1 cells was increased by repetitive exposure to virus such that it was possible to transduce approximately 80% of the cells following three successive daily exposures. The long-term stability of expression of the non-selected IL-2R gene was also evaluated. A slow decline in the percentage of cells expressing IL-2R was seen with cells that were maintained under constant selection pressure for expression of theneorgene, which was expressed from an internal promoter. Simliar results were obtained when cultures were selected initially forneorgene expression and maintained without selection thereafter. In contrast, stable expression was observed in CFT1 cells for at ieast one year following multiple infections in the absence of G418 selection. In conclusion, (i) transduction of foreign genes into human airway epithelial cells using an amphotropic retrovirus vector can be highly efficient in the presence of appropriate polycations and multiple exposures; and (ii) stable expression of a non-selected gene in these epithelial cells is better maintained without selection.