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.
