Prasad Tekkatte Krishnamurthy, Rao Nalam Madhusudhana
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad (AP), India 500007.
Cell Mol Biol Lett. 2005;10(2):203-15.
One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Exp. Cell Res. 230 (1997) 293). In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. For this study, we used transient reporter gene expression assays on various cell types. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids.
在非病毒基因递送中，限制转染效率的步骤之一是质粒DNA一旦被递送至细胞质后，其核输入效率低下。最近，通过向细胞质中显微注射以及对几种细胞类型进行原位杂交，研究表明，猿猴病毒40（SV40）的一个区域，具体而言是包含SV40启动子-增强子-复制起点（SV40 DTS）的约372碱基对的SV40基因组DNA片段，能够使携带这些序列的质粒发生核输入（迪恩·D·A. 《实验细胞研究》230（1997）293）。在本报告中，我们探讨了SV40 DTS用于阳离子脂质介导的基因递送的适用性问题，以及其提高转染过程效率的能力。在本研究中，我们对多种细胞类型进行了瞬时报告基因表达分析。携带SV40 DTS的质粒构建体的基因表达随细胞类型和所用质粒构建体的不同而变化。这种细胞类型和质粒构建体对含有SV40 DTS的质粒基因表达的依赖性表明，质粒的基因表达并不完全依赖于其增强所述质粒核输入的能力。
