The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Abstract

Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.