电穿孔法介导醛脱氢酶基因与多药耐药基因的转移和表达

Electroporation-Mediated Gene Transduction and Expression of Class1Aldehyde Dehydrogenase(ALDH1)and Multidrug Resistance Gene(MDR1)

背景与目的:将不同类型的耐药基因同时导入造血细胞,以扩大耐药范围和进行体内选择是基因治疗的重要策略,这类载体基因片段较长,进行基因转移有一定的难度。本研究旨在寻找安全有效的长片段基因转移途径。方法:将携带不同类型的耐药基因醛脱氢酶基因(ALDH1)和多药耐药基因(MDR1)的逆转录病毒载体G1Na-AIM以NdeI酶切线性化,电穿孔法转导PA317细胞,经长春新碱(VCR)和4-氢过氧化环磷酰胺(4-HC)选择后,应用Southernblot法确定原病毒的整合,逆转录聚合酶链反应(RT-PCR)和流式细胞术(FCM)分析转移基因的表达,筑巢式聚合酶链反应(nestedPCR)检验转移系统的安全性。结果:电穿孔法有效介导了ALDH1与MDR1基因的同时转移,Southernblot法证实ALDH1与MDR1基因稳定整合至宿主细胞基因组,RT-PCR检测到转移基因的转录,FCM测定下游基因MDR1蛋白表达增高4倍,转导率达98%。nestedPCR未检测到辅助病毒(env)。结论:电穿孔法安全有效地介导了ALDH1与MDR1基因的共转移和高表达。

Bcakground &Objective:This study was designed to seek an eff icient and safe method for transfer o f genes of large size.Methods:The retroviral vector containing di fferent kinds drug resistance genes-class1aldehyde dehydrogenase(ALDH1)and multidrug resistance gene(MDR1)was linearized by the restriction en zyme NdeI digestion,and introduced into the packaging PA317cells by eletroporation.Selection was performed by vincristine(VCR)and4-hydroperoxycyclophosphamide(4-HC)to obtain ALDH1and MDR1stably expre ssing cells.The integration of provirus,transcription and translation of fo reign genes were confirmed by Southern blot,reverse transcription(RT)-PCR and flow cytometry,respectively.The safety of this delivery system was verifie d by testing helper virus(envelop gene)using nested PCR.Results:Both ALDH1and MDR1were successfull y transduced into PA317cells by electroporation.Stable integration of foreign genes in host cells genome was determined by Southern hybridization blot.The transcript ion of ALDH1and MDR1was demonstrated by RT-PCR.The overexp ression of P-glycoprotein encoded by the downstream gene MDR1w ith approximately a 4-fold increase in 98%cells was analyzed by flow cytometry.No helper vir us can be detected by nested PCR assay.Conclusion:These results implicate that the int roduction and overexpression of both ALDH1and MDR1genes in vitro is attainable by a simple and convenien t electroporation method,with the c haracter of safety and high efficien cy.