Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates.
Although coprecipitates of plasmid DNA and calcium phosphate (DNA:CaPi coprecipitates) were one of the first methods used for transfection of mammalian cells, they are inefficient for many cell types. Based on the recent finding that incorporating recombinant adenovirus in CaPi coprecipitates enhances expression of virus-encoded transgenes, we tested the hypothesis that including adenovirus in DNA:CaPi coprecipitates would increase the efficiency of transfection. We found that including adenovirus at the time of coprecipitate formation increased transgene expression in several cell types. Only a short incubation with cells was required, and the coprecipitates could be delivered in the presence of serum. Inclusion of adenovirus in coprecipitates did not increase DNA uptake by cells, and inactivated virus was also effective. Neutralizing anti-hexon antibody attenuated the enhancement produced by incorporating virus. These data suggest that the virus enhanced expression at a step after cellular uptake, probably by increasing DNA release from endosomes. The DNA:CaPi:Ad coprecipitates were at least as effective as complexes of DNA with adenovirus and polethylenimine or Lipofectin, but produced less cellular toxicity. The results suggest that DNA:CaPi:Ad coprecipitates have advantages for in vitro gene transfer and provide an attractive vehicle for investigating cellular mechanisms of gene transfer.
Lee
J H
JH
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
Welsh
M J
MJ
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002130:Calcium Phosphates; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D010957:Plasmids; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D002130:Calcium Phosphates; D008156:Luciferases; D001616:beta-Galactosidase