Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells.

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.

Coexpression of 16.8 kDa antigen of Mycobacterium avium paratuberculosis and murine gamma interferon in a bicistronic vector and studies on its potential as DNA vaccine.

Paratuberculosis or Johne's disease is a chronic gastric disease of ruminants. For this disease there is no effective treatment or preventive measure available. 16.8 kDa protein is an immunogenic protein of Mycobacterium avium paratuberculosis and can be an ideal candidate for developing a DNA vaccine construct. In present study a bicistronic DNA vaccine construct pIR16.8/IFN was developed using eukaryotic vector pIRES 6.1. Two genes MPT (expressing 16.8 kDa protein) and murine IFNgamma were cloned, expressed and immunoreactivity was studied in murine model. Immunoreactivity was also compared with monocistronic construct pIR16.8 expressing 16.8 kDa protein. Both pIR16.8 and pIR16.8/IFN showed eukaryotic expression of respective proteins in BHK21 cells. The expressed proteins also showed immunoreactivity when reacted with hyperimmune sera raised against recombinant 16.8 kDa protein in western blot assay and immunofluorence assay. Both constructs were used as DNA vaccine in murine model and immunogenecity was studied by DTH, lymphocyte proliferation assay and NO determination. DTH reaction was significantly high in pIR16.8/IFN than pIR16.8 group, similarly lymphocyte proliferation and NO release was higher in pIR16.8/IFN group than pIR16.8 group. This indicated T cell epitopic nature of 16.8 kDa protein. The study also showed that co-expression of IFNgamma with mycobacterial gene can enhance immunogenecity of DNA vaccine and can be used as immunoadjuvant.