Viral vector-mediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. We compared multiple commercially available transfection reagents to determine which was optimal for retroviral infections of primary myoblasts. Centrifugation force was also tested, and a spin infection protocol with centrifugation at 2800 × g for 90 min had the highest infection efficiency for primary myoblasts. We confirmed that infected muscle stem cells maintain cell proliferation and the capacity for in vitro and in vivo myogenic differentiation. Our new, efficient retroviral infection protocol for muscle stem cells can be applied to molecular biology experiments as well as translational studies.
Bibliographical noteFunding Information:
We thank Conor Burke-Smith for critical reading. This work was supported by the NIH R01 (1R01AR062142) and NIH R21 (1R21AR070319). This paper is subject to the NIH Public Access Policy.
© 2017, Eaton Publishing Company. All rights reserved.
- Muscle stem cell
- Retroviral vector
- Satellite cell
- Skeletal muscle