ABSTRACT
Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.
Subject(s)
Fibroblasts/metabolism , Goats/genetics , Myostatin/genetics , RNA, Small Interfering/metabolism , Transfection , Animals , Base Sequence , Cell Count , Cell Proliferation , Cell Survival , Fibroblasts/cytology , Gene Expression Regulation , Gene Silencing , Molecular Sequence Data , Myostatin/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Time FactorsABSTRACT
Myostatin (MSTN), a member of transforming growth factor-beta superfamily is a negative regulator of the skeletal muscle growth. It suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. In the present investigation silencing of MSTN gene in caprine fibroblast cell line was evaluated using four different shRNA expressing constructs. Variation in the efficiency of silencing (22-92%) was obtained among different constructs. It was observed that sh1 and sh4 constructs downregulated the MSTN gene expression by reducing 92.4 and 80.5% (P<0.05) level of downregulation MSTN mRNA, respectively. On the contrary, the sh3 construct significantly upregulated the MSTN mRNA level (P<0.05). These two promising constructs (sh1 and sh4) need to be further tested for interferon (IFN) response before their use in long term stable expression of anti-MSTN shRNA in muscle cells to improve chevon production.
