PURPOSE: We tried to assess the optimal conditions to improve low transduction efficiency and their effect on target cells. METHODS: Cultured NIH 3T3 cells were incubated with retroviral vectors bearing an enhanced green fluorescent protein (eGFP) gene. We varied the ratio of viral vectors to target cells (1:1-1:8) and the number of transfections (x1, x2), and compared transduction efficiencies. Also, the effects of polybrene on transduction efficiency and viability of target cells were assessed. Transduction of the eGFP gene was evaluated by observing NIH 3T3 cells under a fluorescence microscope and efficiencies were measured by the percentage of eGFP positive cells using FACscan. RESULTS: As the ratio of retroviral vectors to target cells increased, transduction efficiency was greatly improved, from 7% (1:1) to 38% (1:4). However, transduction efficiency did not increase any more when the ratio increased from 1:4 to 1:8. Cells transfected twice showed higher transduction efficiencies than cells transfected once, at a ratio of 1:8. The eGFP gene transduced to NIH 3T3 cells sustained its expression during repeated passages. However, after the third passage (day 9), the percentage of eGFP positive cells began to decline. The degree of this decline in eGFP expression was lower in cells transfected twice than in cells transfected once (P<0.05). The addition of polybrene did not have any toxic effect on NIH 3T3 cells and greatly increased transduction efficiency (P=0.007). In addition to vector component, transduction efficiency was very sensitive to culture confluence. Cells cultured and transfected in 24-well plate showed higher transduction efficiency, although cells cultured in 6- well plate proliferated more (P=0.024). CONCLUSION: Our data could be used as a basis for retrovirus-based gene therapy. Further study will follow using human cells as target cells.
Fluorescence ; Genetic Therapy ; Hexadimethrine Bromide ; Humans ; NIH 3T3 Cells* ; Retroviridae ; Transfection ; Zidovudine

BACKGROUND: Transplantation of cardiac myocytes (CMs) into the injured heart emerges as a potential alternative for the treatment of heart failure. Genetic modification of CMs could enhance and/or modify its therapeutic effects. The characteristics of retroviral gene delivery, which is most commonly used in human trial, has been minimally studied in CMs due to its low efficiency in non-dividing cells. In this study, using newly developed high-titer retrovirus, we evaluated 1) the efficiency of gene transfer into CMs, 2) whether S phase during infection is necessary for the transduction, and 3) characteristics of gene delivery to mononucleated vs binucleated CMs. METHODS: Enriched CMs were cultured from the ventricles of 1 day-old rat hearts. The cells were transduced by MFG-nls-LacZ retroviruses (5x107 IU/ml) in the presence or absence of polybrene. 3H-thymidine was added to label cells in S phase. The cells were stained for b-galactosidase activity and then immunostained using MF20Ab to identify CMs. The cells were subsequently processed for in vitro autoradiography. RESULTS: 1)With 3 rounds of infection, 5.9% of total cultured cells were LacZ-positive. The efficiency of transduction reached upto 7.4% in the presence of polybrene 8 microgram/ml. 2)Nuclear morphology of LacZ-positive CMs was pleomorphic from mononucleated to multinucleated, mostly binucleated. 3)Among mononucleated CMs, 17% of cells were labelled with thymidine. Transduction efficiency (TDE) of thymidine-positive and -negative mononucleated CMs were 37.9% and 3.1%, respectively. Among binucleated cells, 28.9% of cells were labelled with thymidine. TDE of thymidine-positive and -negative binucleated CMs were 75.4% and 13.6%, respectively. 4)In total, TDE of binucleated cells were 3.5 times compared to the one of mononucleated cells (31.5% vs 9.0%). CONCLUSION: TDE of CMs using high-titer retrovirus is relatively low. S phase of cells during retroviral infection is not mandatory for the retroviral transduction. Binucleated CMs are susceptible to retroviral gene delivery and their TDE is higher than the one of mononucleated CMs.
Animals ; Autoradiography ; Cells, Cultured ; Dichlorodiphenyldichloroethane ; Genetic Therapy ; Heart ; Heart Failure ; Hexadimethrine Bromide ; Humans ; Myocytes, Cardiac* ; Rats ; Retroviridae* ; S Phase ; Thymidine ; Zidovudine