逆转录病毒载体介导的耐药基因在造血细胞中基因转导和表达的研究

Study on Transduction and Expression of Drug Resistance Gene Mediated by Retrovirus Vector in Hematopoietic Cells

本文对于逆转录病毒载体介导的基因转移方法中如何提高逆转录病毒滴度进行了研究。以磷酸钙共沉淀的方法转染包装细胞PA17,经氨甲蝶呤(MTX)10^(-7)mol/L筛选,扩增得到产病毒细胞株,经点杂交证实dhfr基因成功导入并产生病毒颗粒。混合细胞克隆PA317/pSPD产生的逆转录病毒滴度为(1.1-2.3)×10~3 cfu/ml。随MTX筛选浓度增加,产病毒细胞PA317/pSPD抗性增加。当培养基中MTX浓度由10^(-7) mol/L增加到10^(-6)mol/L时,逆转录病毒滴度增加了近10倍[(1.02-2.09)×10~4 cfu/ml]。结果显示可以通过增加MTX筛选浓度来提高含dhfr cDNA的逆转录病毒滴度。

Retrovirus vector pSPD mediated gene transfer was applied in this study in which a mutant dihy-drofolate reductase gene was transduced. Calcium phosphate coprecipitation was used to transfect PA317 packaging cell line. Dot blot confirmed the successful transduction of dhfr gene and production of virions. Our data show that the dhfr-virus titer could be increased about 10-fold when increasing MTX concentration from 10 -7 mol/L to 10 -6 mol/L. These results demonstrated the potential for increasing the dhfr virus titer by selection in increasing MTX concentration. It is suggested to be a useful method to increase the concentration of target gene-containing virus when dhfr gene being used as a marker gene.