Polyoma virus pseudocapsids as efficient carriers of heterologous DNA into mammalian cells.
Polyoma virus VP1 pseudocapsids, generated from a recombinant baculovirus, have been successfully used to transfer exogenous DNA stably into rodent (rat-2) cells. To evaluate the efficiency and biological usefulness of this route for introducing heterologous DNA into cells, the gene for a transforming deletion mutant of the middle T antigen of polyoma virus, dl8 MT, was used initially. Whereas the amount of DNA packaged together with pseudocapsids was found to be variable (2-30%), even at low efficiency its transfer as biologically functional information was high. The dl8 MT gene was stably transferred and integrated in low copy numbers into the host chromosome. Transformed cell lines (derived from single foci) were shown to produce high levels of the corresponding mutant protein, which was active in an in vitro protein kinase assay. In comparisons with the calcium phosphate DNA coprecipitation procedure (or lipofectin route), the VP1 pseudocapsid approach was shown to have many advantages in terms of maintenance of DNA fidelity and increased efficiency of gene expression. This system was also assessed for its ability to transfer into and express the chloramphenicol acetyl transferase (CAT) gene in a human liver cell line. Here again, the assay for functional CAT expression showed the pseudocapsid transfer procedure to compare favorably with lipofectin transfer. In another transient assay, a low-level endogenously expressed gene, p43, was complexed with pseudocapsids and transferred into human embryo lung fibroblasts, thereby increasing the expression levels. The ease of production of VP1 pseudocapsids, coupled with their efficient transfer of biologically useful information, should make this route of gene delivery an attractive proposition for further exploration with regard to gene therapy.
Forstová
J
J
Department of Virology, Royal Postgraduate Medical School, London, UK.
Krauzewicz
N
N
Sandig
V
V
Elliott
J
J
Palková
Z
Z
Strauss
M
M
Griffin
B E
BE
D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics; D002213:Capsid / Q000235:genetics*; D002478:Cells, Cultured / Q000821:virology; D004247:DNA / Q000235:genetics*; D018014:Gene Transfer Techniques; D006087:Graft vs Host Reaction; D006801:Humans; D008322:Mammals / Q000235:genetics* / Q000821:virology*; D011120:Polyomavirus / Q000235:genetics*; D051381:Rats
D000956:Antigens, Viral; D004247:DNA