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J Thorac Cardiovasc Surg 1998;115:1-8
© 1998 Mosby, Inc.

CARDIAC AND PULMONARY REPLACEMENT

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts

Supported by The British Heart Foundation (PG/96085).

Read at the Seventy-seventh Annual Meeting of The American Association for Thoracic Surgery, Washington, D.C., May 4-7, 1997.

Received for publication May 7, 1997; revisions requested July 11, 1997; revisions received August 7, 1997; accepted for publication August 8, 1997. Address for reprints: Reida El Oakley, FRCS, MD, Imperial College School of Medicine at The National Heart and Lung Institute, Dovehouse St., London SW3 6LY, United Kingdom.

Abstract

Background: Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose.
Methods: MFGnlslacZ retroviral vector, packaged in high-titer, split-genome packaging cell line (FLYA4) was used to transduce the skeletal myoblast cell line L6. L6 cells, cultured in 10% fetal calf serum, were transduced with the MFGnlslacZ vector by means of filtered supernatant from FLYA4 cells. Transduced L6 cells were divided into four groups. Group I cells were fixed as myoblasts 3 days after transduction. Group II cells were allowed to differentiate into myotubes. Group III cells were split every 3 days for 4 months. Group IV cells were split as in group III but then allowed to differentiate into myotubes. All samples were fixed and stained for ß-galactosidase activity. The effects on gene transfer of transforming growth factor–ß, insulin-like growth factor–I, and platelet-derived growth factor were determined by spectrophotometric assay of ß-galactosidase activity in cells transduced in the presence or absence of serum with 0 to 200 ng/ml of each growth factor.
Results: Morphometric analysis showed that 66.3% ± 3% to 69.6% ± 6% of cells in groups I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor–ß significantly inhibited gene transfer, whereas insulin-like growth factor–I and platelet-derived growth factor significantly enhanced gene transfer. In absence of serum, however, only platelet-derived growth factor enhanced retrovirally mediated gene transfer into skeletal myoblasts.
Conclusion: MFG retroviral vectors packaged in FLYA4 cells are efficient in gene transfer into skeletal myoblasts and result in transgenic expression that is maintained after repeated cell division, differentiation, or both. Platelet-derived growth factor enhances retrovirally mediated gene transfer into skeletal myoblasts.

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				R. M. El Oakley, O. C. Ooi, A. Bongso, and M. H. Yacoub Myocyte transplantation for myocardial repair: a few good cells can mend a broken heart Ann. Thorac. Surg., May 1, 2001; 71(5): 1724 - 1733. [Abstract] [Full Text] [PDF]