IN VITRO MYOSTATIN GENE SILENCING BY shRNA IN CHICKEN EMBRYO MYOBLAST CELLS

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dc.contributor.advisorJoshi, Chaitanya G.
dc.contributor.authorTRIPATHI, AJAI KUMAR
dc.date.accessioned2018-04-27T08:40:20Z
dc.date.available2018-04-27T08:40:20Z
dc.date.issued2012
dc.description.abstractMyostatin (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<O.Ol) in compare to mock transfected control. Top five shRNA constructs were selected for myostatin gene silencing in chicken embryo myoblast cells, which were cultured from 11-12 days old WLH chicken embryo pectoraUs major (breast) muscle. Myoblast cells were isolated from this muscle, using 20/60% percoll gradient. After collection, cells were washed twice with MEM-a medium and plated @ of 2.5 x 105 cells per well in a 12-well plate in complete growth medium. Chicken myoblast cells (80-90% confluence) were transfected in triplicates with selected shRNAs with suitable controls. The cells exhibited only 22.56±0.41% transfection efficiency. RT-qPCR analysis of transfected myoblast cells showed up to 250% myostatin gene silencing (sh_4). Moreover, transfection of chicken myoblast cells also induced OASl and IFNfi expression (2.1 to 93.6 folds) compare to mock transfected cells (p<0.01). In view of side effects of RNAi, it becomes increasingly important to test all constructs not only for high RNAi efficiency but also for induction of IFN response. However, to increase the muscle mass in the transgenic animals, it will require long term stable expression of anti-myostatin shRNA. This requires the development of stably transfected (anti-myostatin shRNA transformed) cell line which may be used as a donor cell for the creation of transgenic chicken with enhanced muscle growth.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/5810044691
dc.keywordsIN VITRO MYOSTATIN GENE SILENCING, shRNA IN CHICKEN EMBRYO MYOBLAST CELLSen_US
dc.language.isoenen_US
dc.publisherAAU, Ananden_US
dc.research.problemIN VITRO MYOSTATIN GENE SILENCING BY shRNA IN CHICKEN EMBRYO MYOBLAST CELLSen_US
dc.subAnimal Biotechnologyen_US
dc.subjectANIMAL BIOTECHNOLOGYen_US
dc.subjectA STUDYen_US
dc.themeIN VITRO MYOSTATIN GENE SILENCING BY shRNA IN CHICKEN EMBRYO MYOBLAST CELLSen_US
dc.these.typePh.Den_US
dc.titleIN VITRO MYOSTATIN GENE SILENCING BY shRNA IN CHICKEN EMBRYO MYOBLAST CELLSen_US
dc.typeThesisen_US
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