Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro

Abstract

Background: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. Methods: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. Results: We observed a significant silencing (64–81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16–46% silencing with maximum of 46% by sh6 (p = 0.0318) against endogenous ACVR2B whereas up to 66% (p = 0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r = 0.547; p = 0.102), myogenin (r = 0.517; p = 0.126) and Myf5 (r = 0.262; p = 0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. Conclusions: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.

• ACVR2B knockdown
• goat myoblasts
• HEK293T cells
• interferon response
• shRNAs