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Goat Activin Receptor Type IIB Knockdown by Artificial MicroRNAs In Vitro

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Abstract

Activin receptor type IIB (ACVR2B) has been known to negatively regulate the muscle growth through mediating the action of transforming growth factor beta superfamily ligands. Recently, the artificial microRNAs (amiRNAs) which are processed by endogenous miRNA processing machinery have been proposed as promising approach for efficient gene knockdown. We evaluated amiRNAs targeting goat ACVR2B in HEK293T and goat myoblasts cells. The amiRNAs were designed based on the miR-155 backbone and cloned in 5′- and 3′-UTR of GFP reporter gene under the CMV promoter. Although both 5′- and 3′-UTR-amiRNAs vectors showed efficient synthesis of GFP transcripts, amiRNAs in 5′-UTR drastically affected GFP protein synthesis in transfected goat myoblast cells. Among the four amiRNAs targeting ACVR2B derived from either 5′- or 3′-UTR, ami318 showed highest silencing efficiency against exogenously co-expressed ACVR2B in both 293T and goat myoblast cells whereas ami204 showed highest silencing efficiency against endogenous ACVR2B in goat myoblasts cells. The 3′-UTR-derived amiRNA exerted higher knockdown efficiency against endogenous ACVR2B at transcript level whereas 5′-UTR-derived amiRNAs exerted higher knockdown efficiency at protein level. The expression of ACVR2B showed positive correlation with the expression of MYOD (r = 0.744; p = 0.009) and MYOG (r = 0.959; p = 0.000) in the amiRNA-transfected myoblasts. Although both 5′- and 3′-UTR–amiRNA vectors led to substantial induction of interferon response, the magnitude of the response was found to be higher with the 3′-UTR–amiRNA vectors.

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Acknowledgments

Authors wish to thank National Agriculture Innovation Project (NAIP), New Delhi, India, and Anand Agricultural University, Gujarat, India, for providing necessary funding to carry out the research work.

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  1. Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, 388001, Gujarat, India

    Amrutlal K. Patel, Ravi K. Shah, Ishan K. Parikh & Chaitanya G. Joshi

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  1. Amrutlal K. Patel
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  2. Ravi K. Shah
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Correspondence to Chaitanya G. Joshi.

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Patel, A.K., Shah, R.K., Parikh, I.K. et al. Goat Activin Receptor Type IIB Knockdown by Artificial MicroRNAs In Vitro. Appl Biochem Biotechnol 174, 424–436 (2014). https://doi.org/10.1007/s12010-014-1071-3

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  • DOI: https://doi.org/10.1007/s12010-014-1071-3

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