title: Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts.
abstract: Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (p = 0.003) and 54% (p = 0.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, p = 0.001), MYOG (94%, p = 0.000), and MYF5 (36%, p = 0.000), cell cycle regulator p21 (62%, p = 0.000), MSTN receptor ACVR2B (23%, p = 0.061), MSTN antagonist follistatin (81%, p = 0.000), and downstream signaling mediators SMAD2 (20%, p = 0.060) and SMAD3 (49%, p = 0.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (p = 0.354) and 79% compared to untransduced myoblasts (p = 0.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; p = 0.000)- and sh2 (1.71-fold; p = 0.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, 388 001, Gujarat, India.
authors.forename: Amrutlal K
authors.initials: AK
authors.lastname: Patel
authors.forename: Ajai K
authors.initials: AK
authors.lastname: Tripathi
authors.forename: Utsav A
authors.initials: UA
authors.lastname: Patel
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Shah
authors.forename: Chaitanya G
authors.initials: CG
authors.lastname: Joshi
mesh_terms: D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism; D030301:Activin Receptors, Type II / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D038681:Follistatin / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006041:Goats; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D017570:MyoD Protein / Q000378:metabolism; D032446:Myoblasts / Q000502:physiology*; D051787:Myogenic Regulatory Factor 5 / Q000378:metabolism; D018008:Myogenin / Q000378:metabolism; D055435:Myostatin / Q000235:genetics*; D053652:Nuclear Transfer Techniques; D034741:RNA, Small Interfering / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism
chemical_list: D017931:DNA Primers; D038681:Follistatin; D017570:MyoD Protein; D051787:Myogenic Regulatory Factor 5; D018008:Myogenin; D055435:Myostatin; D034741:RNA, Small Interfering; D051899:Smad2 Protein; D051900:Smad3 Protein; D030301:Activin Receptors, Type II; D015088:2',5'-Oligoadenylate Synthetase