Lewis E, Rudo T, St John M R, Chu J, Heinze A, Howard B, Engleka K
Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107-6799, USA.
Gene Ther. 1999 Sep;6(9):1617-25. doi: 10.1038/sj.gt.3300977.
The nonreplicating vaccinia virus MVA/T7 RNA polymerase hybrid system was tested with Petri dish electroporation for ectopic gene expression in human umbilical vein endothelial cells (HUVECs). A range of voltages (150-450 V), pulse times (10-40 ms), DNA concentrations (0-20 microg/ml) and infection levels (0-15 multiplicities of infection) were tested for effects on T7 promoter-directed chloramphenicol acetyltransferase (CAT) activity after MVA/T7RP infection. MVA/T7RP-directed expression was transient and at least 10 000-fold in excess of nonviral, cytomegalovirus enhancer-directed expression. Use of a Petri dish electrode with the MVA/T7RP system showed increased viability compared with a cuvette electrode. Overexpression of interleukin-2 alpha subunit (IL2Ralpha) pro- tein followed by anti-IL2Ralpha-directed magnetic immunoaffinity cell sorting allowed isolation of the transfected population. The high fidelity of cellular sorting was shown by segregation of CAT activity in the IL2Ralpha-sorted population after transfection of T7 promoter-directed bicistronic IL2Ralpha/CAT DNA. Expression of a panel of proteins including the fluorophore green fluorescent protein as detected by fluorescence microscopy and p21cip1, p27kip1, pp60c-src, FGF-1, pRb, p107 and pRb2/p130 proteins was also achieved. Thus, use of the nonreplicating vaccinia virus/T7 RNA polymerase expression system with Petri dish electroporation is feasible for certain applications for the manipulation of HUVECs by gene transfer.
采用培养皿电穿孔法，利用非复制型痘苗病毒MVA/T7 RNA聚合酶杂交系统在人脐静脉内皮细胞（HUVECs）中进行异位基因表达检测。检测了一系列电压（150 - 450 V）、脉冲时间（10 - 40 ms）、DNA浓度（0 - 20 μg/ml）和感染水平（0 - 15感染复数）对MVA/T7RP感染后T7启动子驱动的氯霉素乙酰转移酶（CAT）活性的影响。MVA/T7RP介导的表达是瞬时的，且比非病毒的巨细胞病毒增强子介导的表达至少高10000倍。与比色杯电极相比，在MVA/T7RP系统中使用培养皿电极显示细胞活力有所提高。通过抗IL2Rα导向的磁性免疫亲和细胞分选，在过表达白细胞介素-2α亚基（IL2Rα）蛋白后，可分离出转染群体。转染T7启动子驱动的双顺反子IL2Rα/CAT DNA后，在IL2Rα分选群体中CAT活性的分离表明细胞分选的高保真度。通过荧光显微镜检测，还实现了包括荧光团绿色荧光蛋白以及p21cip1、p27kip1、pp60c-src、FGF-1、pRb、p107和pRb2/p130蛋白在内的一组蛋白质的表达。因此，将非复制型痘苗病毒/T7 RNA聚合酶表达系统与培养皿电穿孔法结合使用，对于通过基因转移操纵HUVECs的某些应用是可行的。
