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Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts
R M el Oakley1, N J Brand, P B Burton
1Department of Cardiothoracic Surgery, Imperial College School of Medicine, National Heart and Lung Institute, London, United Kingdom.
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 beta-galactosidase activity. The effects on gene transfer of transforming growth factor-beta, insulin-like growth factor-I, and platelet-derived growth factor were determined by spectrophotometric assay of beta-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 group I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor-beta 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.