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Anand Agricultural University, Anand

Anand Agricultural University (AAU) was established in 2004 at Anand with the support of the Government of Gujarat, Act No.(Guj 5 of 2004) dated April 29, 2004. Caved out of the erstwhile Gujarat Agricultural University (GAU), the dream institution of Sardar Vallabhbhai Patel and Dr. K. M. Munshi, the AAU was set up to provide support to the farming community in three facets namely education, research and extension activities in Agriculture, Horticulture Engineering, product Processing and Home Science. At present there seven Colleges, seventeen Research Centers and six Extension Education Institute working in nine districts of Gujarat namely Ahmedabad, Anand, Dahod, Kheda, Panchmahal, Vadodara, Mahisagar, Botad and Chhotaudepur AAU's activities have expanded to span newer commodity sectors such as soil health card, bio-diesel, medicinal plants apart from the mandatory ones like rice, maize, tobacco, vegetable crops, fruit crops, forage crops, animal breeding, nutrition and dairy products etc. the core of AAU's operating philosophy however, continues to create the partnership between the rural people and committed academic as the basic for sustainable rural development. In pursuing its various programmes AAU's overall mission is to promote sustainable growth and economic independence in rural society. AAU aims to do this through education, research and extension education. Thus, AAU works towards the empowerment of the farmers.

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20121
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Subject: Animal Biotechnology × Author: TRIPATHI, AJAI KUMAR × Start date: 1997 × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    IN VITRO MYOSTATIN GENE SILENCING BY shRNA IN CHICKEN EMBRYO MYOBLAST CELLS
    (AAU, Anand, 2012) TRIPATHI, AJAI KUMAR; Joshi, Chaitanya G.
    Myostatin (MSTN), a member of transforming growth factor-P (TGF-P) superfamily, is a negative regulator of the skeletal muscle growth as it suppresses the proliferation and differentiation of myoblast cells. Scientists have reported that mice genetically engineered to lack myostatin activity have about twice the amount of muscle mass throughout the body. Dysfunction of MSTN either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the muscle mass in manmialian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN knockdown by developing short hairpin RNA (shRNA) construct against it. It is considered that in vitro knockdown of MSTN gene in chicken embryo myoblast by shRNA expressing constructs would help in devising suitable in vivo strategies for MSTN gene knockdown. This, in turn, might help to produce transgenic chicken with increased muscle mass. Apart from meat quantity, the quality of meat will also be improved as the knock down of myostatin gene will result in more lean type of meat which is advisable for the safe and healthy meat consumption. In the present study, shRNA induced myostatin gene silencing in in vitro chicken myoblast culture was evaluated using seven different shRNA expressing constructs by quantitative Real Time PCR. Myostatin silencing efficiency of shRNA constructs was first evaluated in Human embryonic kidney cell-line 293T (HEK 293T) cells, which are frequently used for its extreme transfectability by the various techniques and exogenously express target proteins. Seven antimyostatin constructs were used in this study, of which six were designed from online tool using GU075928 sequence as input, and one siRNA was selected form literature. The antisense strands were checked for the presence of any secondary structure. Out of seven; one construct formed secondary structures in its antisense strand, whereas no secondary structures were found in rest six constructs. These constructs were cloned into pSIREN vector between EcoRI and BamHl restriction sites. Sequencing results of shRNA constructs revealed no mutation in any of the shRNA constructs. Full length chicken myostatin gene was amplified from total RNA extracted from 11-12 days WLH chicken embryo, and cloned in to pcDNA3 expression vector and synthesized pcDNA3_cMSTN constructs. This expression vector was cotransfected with each shRNA into HEK293T cells. HEK 293T cells were seeded 2.5 x 10 power of 5 cells per well in a 12-well plate, and transfections were performed in triplicates. Transfection efficiency of the combinations did not vary much (80.05±0.73%). Total RNA was extracted from transfected cells after 36 h of transfection. First strand cDNA was synthesied and qPCR was performed using SYBR Green master mix, gene specific primers for GAPDH (endogenous control) and MSTN, OASl, IFNp and PKR (targets); and cDNA as template. Amplified PCR products were electrophoresed on 2% agarose, which revealed single compact band of 257 bp in GAPDH, 245 bp in MSTN, 209 bp in OASl, 159 bp in IFN-p and 165 bp in PKR. These shRNA constructs were having efficient myostatin silencing effect, ranging from 37.5% to 95.6%. The induction of interferon genes {OASl, IFN J3 and PKR) expression was significant (1.1 to 23.5 folds, p