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Detection Of FST Transgenic Sheeps

Posted on:2016-04-20Degree:MasterType:Thesis
Country:ChinaCandidate:K WangFull Text:PDF
GTID:2180330461982382Subject:Zoology
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Since the first genetically engineered or transgenic mice was producted in 1980s. A variety of transgenic technologies have been used successfully to produce transgenic mice and other transgenic animal species. In recent years,China has made great break-through in the field of transgenic animal research,these results of transgenic techniques can provide a better platform to develop transgenic animals for breeding new animal varieties and livestock production. Our research group used the combination of vector gene transfer and somatic cell nuclear transfer to produce transgenic sheeps which overexpress follistatin(FST) in muscle tissues in 2011.This dissertation makes a research on gene expression、muscle development and animal health of transgenic animal by PCR、southern blot、western blot、real-time quantitative fluorescent PCR technique、muscle tissue sections and blood biochemical analysis to analyze the FST transgenic Mongolian sheeps (NO.14、NO.17、NO.18) and transgenic goat (NO.21).1) Detection of positive transgenic sheeps.The first, PCR and Southern blot were simultaneously used to detect positive transgenic sheeps, then PCR was used to identify exogenous gene constructs. The results showed that:four samples which were detected are all positive, and multiple sites of exogenous gene (SP promoter, a-actin promoter, FST,CMV promoter, DsRed and neomycin gene) had been integrated into the transgenic animals genome.2) Detection of exogenous gene copy number The sheeps endogenous gene glucagon served as an internal control to calculate exogenous gene copy number. Using real-time quantitative PCR, the standard curve was established between copy number and ΔCt. The standard curve log5N=-0.415Δ Ct+0.0194 (R=0.9911) was determined to assay exogenous gene copy number, the results showed that exogenous gene copy number of samples (NO.14, NO.17. NO.18 and NO.21) were 4,10.3 and 1 respectively.3) Analysis of FST, MSTN and BMP4 gene expression in transgenic sheepsIn this experiment, the relative expression level of FST, MSTN. BMP4 gene mRNA were calculated.by real-time quantitative fluorescent PCR technique.The relative expression level of FST gene mRNA in muscle of sheep samples NO.14. NO.17. NO.18 and goat sanple NO.21 were 1.157,1.125,1.021 and 1.959 times respectively, the relative expression level of MSTN gene mRNA in muscle were 1.102.1.804,2.235 and 1.495 times respectively, the relative expression level of BMP4 gene mRNA in muscle were 0.504,0.135,0.165 and 0.265 times respectively; The relative expression level of FST gene mRNA in skin of samples (NO.14, NO.17. NO.18 and NO.21)were 0.868,1.101,1.089 and 1.156 times respectively,the relative expression level of MSTN gene mRNA in skin were 1.172,1.134,1.203 and 1.230 times respectively,the relative expression level of BMP4 gene mRNA in skin were 0.978,1.021,1.142 and 0.451 times respectively.The relative expression level about FST gene protein of sheep samples NO.14. NO.17, NO.18 and goat sanple NO.21 were 1.05、1.13、1.05 and 1.72 times respectively, the relative expression of MSTN gene protein were 0.81、0.70、0.76 and 0.69 times respectively.4) FST transgenic sheep individual-level detection and analysisThe muscle fiber density and fiber diameter of three Mongolia transgenic sheeps (NO.14、NO.17、NO.18) and a transgenic goat (NO.21) were measured with tissue biopsies and HE staining. It was found that muscle fiber density of transgenic Mongolian sheep samples NO.14、NO.17、NO.18 were 246mm2、235mm2、233mm2 respectively, the muscle fiber density of transgenic Mongolian sheep greater than that of control.The muscle fiber density of transgenic goat sample NO.21 was 271mm2 respectively, also larger than normal goat muscle fiber density (224mm2).The muscle fiber diameters of transgenic Mongolian sheep samples (NO.14、NO.17、NO.18) were 55.38μm,64.32μm,58.93μm respectively,the muscle fiber diameter of transgenic sheeps are larger than that of control.The muscle fiber diameter of transgenic goat NO.21 was 62.58μm, higher than that of control. In addition, the transgenic sheeps blood biochemical analysis including liver function, renal function, blood glucose and lipid analysis. The results shows that transgenic Mongolian sheeps and transgenic goat was no significant difference with ordinary non-transgenic sheep and non-transgenic goatThese results show that high expression of FST gene in muscle inhibits the expression of MSTN and BMP4 protein, thus promoting muscle growth and development.The results of this article lay the foundation for future study on the transgenic animals.
Keywords/Search Tags:Genetically modified goat, Real-time PCR, copy number, Southern blot, Western blot, FST
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