Fusigenic liposome-mediated DNA transfer into cardiac myocytes

Kristin E. Ellison, Nanette Bishopric, Keith A Webster, Ryuichi Morishita, Gary H. Gibbons, Yasufumi Kaneda, Barbara Sato, Victor J. Dzau

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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 β-galactosidase (pActβ-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%) 1h 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 pActβ-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 β-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.

Original languageEnglish
Pages (from-to)1385-1399
Number of pages15
JournalJournal of Molecular and Cellular Cardiology
Volume28
Issue number7
DOIs
StatePublished - Jul 1 1996
Externally publishedYes

Fingerprint

Sendai virus
Cardiac Myocytes
Liposomes
Transfection
DNA
Plasmids
Galactosidases
Muscle Cells
Oligonucleotides
Polyomavirus Transforming Antigens
Viral Tumor Antigens
Capsid Proteins
Nuclear Proteins
Fluorescein
Genes
Heart Diseases
Cell Culture Techniques
Fluorescence
Immunohistochemistry
Rabbits

Keywords

  • β-galactosidase
  • Cardiac myocytes
  • Cell culture
  • Gene therapy
  • Gene transfer
  • Liposomes
  • Oligodeoxynucleotides
  • Sendai virus

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Cite this

Ellison, K. E., Bishopric, N., Webster, K. A., Morishita, R., Gibbons, G. H., Kaneda, Y., ... Dzau, V. J. (1996). Fusigenic liposome-mediated DNA transfer into cardiac myocytes. Journal of Molecular and Cellular Cardiology, 28(7), 1385-1399. https://doi.org/10.1006/jmcc.1996.0130

Fusigenic liposome-mediated DNA transfer into cardiac myocytes. / Ellison, Kristin E.; Bishopric, Nanette; Webster, Keith A; Morishita, Ryuichi; Gibbons, Gary H.; Kaneda, Yasufumi; Sato, Barbara; Dzau, Victor J.

In: Journal of Molecular and Cellular Cardiology, Vol. 28, No. 7, 01.07.1996, p. 1385-1399.

Research output: Contribution to journalArticle

Ellison, KE, Bishopric, N, Webster, KA, Morishita, R, Gibbons, GH, Kaneda, Y, Sato, B & Dzau, VJ 1996, 'Fusigenic liposome-mediated DNA transfer into cardiac myocytes', Journal of Molecular and Cellular Cardiology, vol. 28, no. 7, pp. 1385-1399. https://doi.org/10.1006/jmcc.1996.0130
Ellison KE, Bishopric N, Webster KA, Morishita R, Gibbons GH, Kaneda Y et al. Fusigenic liposome-mediated DNA transfer into cardiac myocytes. Journal of Molecular and Cellular Cardiology. 1996 Jul 1;28(7):1385-1399. https://doi.org/10.1006/jmcc.1996.0130
Ellison, Kristin E. ; Bishopric, Nanette ; Webster, Keith A ; Morishita, Ryuichi ; Gibbons, Gary H. ; Kaneda, Yasufumi ; Sato, Barbara ; Dzau, Victor J. / Fusigenic liposome-mediated DNA transfer into cardiac myocytes. In: Journal of Molecular and Cellular Cardiology. 1996 ; Vol. 28, No. 7. pp. 1385-1399.
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abstract = "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 β-galactosidase (pActβ-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{\%}) 1h 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 pActβ-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 β-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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