Key result
HVJ-liposomes achieve up to ~90% oligonucleotide transfection efficiency in cultured cardiac myocytes.
Why the study?
Current methods of gene transfer into cultured cardiac myocytes have serious limitations including low efficiency, toxicity, or constraints on DNA insert size.
HVJ/liposome-mediated transfer is an efficient method for transfecting oligonucleotides and plasmids into cardiac myocytes both in vitro and in vivo, providing a potential new tool for investigating cardiac myocyte biology.
May enable efficient cardiac myocyte transfection studies; leaves open in vivo translation and therapeutic application.
Current methods of gene transfer into cultured cardiac myocytes have serious limitations, including low efficiency, toxicity or constraints on DNA insert size. The present study examined the effectiveness of hemagglutinating virus of Japan (HVJ) in promoting liposome-mediated DNA transfer into cultured neonatal rat cardiac myocytes. Fluorescein isothiocyanate-labeled oligonucleotides (F-ODN) or plasmid expression vectors encoding SV40 large T antigen (pActSVT) and beta-galactosidase (pAct beta-gal) were complexed with liposomes and the viral protein coat of HVJ. Plasmid vectors were complexed with the nuclear localizing protein HMG-1 prior to HVJ-liposome encapsulation. Neonatal myocytes were transfected by incubation with HVJ-liposome/DNA complexes on culture day 3 or 7. Using F-ODN, we were able to demonstrate significant uptake of DNA (transfection efficiencies of 80-90%) 1 h after transfection that persisted for 1 week in culture. Interestingly, F-ODN were concentrated in the myocyte nuclei for the first 4 days after transfection. Immunohistochemistry showed that 25-30% of myocytes transfected with either pActSVT or pAct beta-Gal expressed plasmid-encoded protein at 72 h whether they were transfected at day 3 or day 7 of culture, while cells transfected with blank vectors did not. Quantitative beta-galactosidase assays confirmed that the use of HVJ significantly enhanced liposome-mediated transfection. Cell toxicity was not apparent. Gene transfer via intracoronary injection also demonstrated the capacity of HVJ to mediate transfection of rabbit cardiac myocytes in vivo, with F-ODN-dependent fluorescence persisting for up to 1 week. We conclude that HVJ/liposome-mediated transfer is efficient for the transfection of both oligonucleotides and plasmids into cardiac myocytes both in vitro and in vivo, and may provide a new tool for the investigation of cardiac myocyte biology and disease.
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Ellison et al. (1996) studied this question. HVJ-liposome-mediated DNA transfer vs. Blank vectors was evaluated on Transfection efficiency and protein expression. HVJ-liposome-mediated DNA transfer achieved 80-90% transfection efficiency for oligonucleotides and 25-30% for plasmids in cultured cardiac myocytes, with expression persisting up to 1 week.
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