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The biological requirements for successful somatic gene
therapy include the ability to efficiently introduce recombinant
genes into the target cells.Two general strategies have been
proven to be useful, including both viral vectors and the
formulated DNA expression vectors. Recombinant viral vectorshave
been less desired in the development of gene tranfer strategies
due to the concerns of safety. In the present study, delivery of
the recombinant genes by the nonvirial strategies utilizing the
polyomavirus (Py) major capsid protein VP1 as a gene carrier was
attempted. The results obtained were compared withthose obtained
by Pseudofect,(a recombinant PyVP1 expressed in baculovirus)
andby the calcium phosphate coprecipitation method. The
polymavirus VP1 pseudocapsids generated from recombinant
baculovirus, have been shown to be able to effectively transfer
the exogenous DNA into several mammalian cell lines in vitro.
Experiments were carried out in thisstudy to investigate the
feasibility of transferring foreign DNA into therecipient host
by PyVP1 purifed from recombinant E.coli,both in vitro and in
vivo. The reporter genes employed were pCMVb, containing the
lacZ gene under thecytomegalovirus promoter, and pPyMT-1,
containing the polyomavirus middle Tantigen. To prepare
the E.coli VP1/DNA complex, purified E.coli VP1 was sonicated,
mixed with CsCl - purified DNA and then incubated with an
appropriate amountof poly-L-lysine (MW30000 - 70000) at room
temperature. The NaCl concentrationwas then adjusted to 35 mM,
followed by sonication before use. The level of DNA
protected by VP1 was first examined by incubating DNAwith VP1,
followed by DNase digestion. The experimental results indicated
that exogenous DNA was not protected by E.coli VP1, whereas
about 5-10% of the inputDNA was protected from DNase treatment
by VP1 Pseudofect. NIH3T3, a polyomavirus permissive cell
line, and FR3T3, a polyomavirustransformation competent cell
line were transfected by pCMVb and pPyMT-1,respectively.In vitro
DNA transfection was carried out by treating the cells withE.
coli VP1,Psesudofect, and the calcium phosphate coprecipitation
methods.Expression of b-galactosidase or PyMT-1 reporter genes
was examined by X-galstaining, exzyme-linked immunosorbent assay
(ELISA), and Western blot followedby PhosphorImager analysis.
The results obtained showed that although expression of b-gal
was detected in several cells at 48 hours post-transfection, the
efficiency of gene transfer by E.coli was about 30-50% as
compared to the calciumphosphate method. VP1 Pseudofect,
generated from baculovirus, on the other hand,showed a lower
transfection efficiency than those reported by others.
Nostatisticall significant difference on the in vitro
transfection efficiency was observed for PyVP1 obtained from two
different sources. Parallel experiments were performed in
vivo by injecting pCMVb or pPyMT-1 plasmid DNA/E.coli VP1
mixture into the livers of Wistar rats. Liverswere harvested 72
hours after direct gent transfer. Expression of the reporter
genes were analyzed by X-gal staining, ELISA, and Western blot
analysis, andthe results were compared with those obtained by
direct injection of naked DNAsolution and DNA-poly-L-lysine
complex suspensions. Direct injection of plasmid DNA into liver
exhibited sufficient gene expression. Complexation withpoly-L-
lysine was found to increase about 1.5 fold of the reporter
geneexpression. Microscopic examination of liver sections
revealed intracellularblue granules consisten with b-gal
activity. The majority of transfected hepatocytes were found to
be localized in the vicinity of the injection sites.Semi-
quantitative analysis of b-gal and PyMT-1 gene expression by
ELISA andWestern blot analysis suggested that PyVP1 obtained
either from baculovirus orE.coli exhibits no significant effect
on the efficiency of gene transfer in vivo.
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