本文選題：Fat-1基因 + 體細胞克隆�。� 參考：《內(nèi)蒙古大學》2015年碩士論文

【摘要】：Fat-1基因編碼的產(chǎn)物是多不飽和脂肪酸脫氫酶,能將n-6多不飽和脂肪酸(PUFAs)轉(zhuǎn)化為n-3 PUFAs。本研究利用MAR (matrix attachment regions)序列作為啟動元件,構(gòu)建了MAR-CAG-Fat-1-PolyA-MAR單順反子表達盒,轉(zhuǎn)染渤海黑牛胎兒成纖維細胞,通過有限稀釋法挑取單克隆細胞進行培養(yǎng),提取基因組DNA進行鑒定,獲得了轉(zhuǎn)基因陽性單克隆細胞系。然后,對轉(zhuǎn)基因陽性細胞DNA整合,RNA、蛋白表達、脂肪酸含量等鑒定分析。用pMAR-CAG-Fat-1-PolyA-MAR載體(單酶切載體)和MAR-CAG-Fat-1-PolyA-MAR載體(雙酶切載體)分別制備轉(zhuǎn)基因小鼠。將表達良好的轉(zhuǎn)基因單克隆細胞通過體細胞核移植技術(shù)制備克隆胚胎。結(jié)果表明,(1)獲得了穩(wěn)定轉(zhuǎn)染雙酶切載體的單克隆陽性細胞系,該細胞系的n-3 PUFAs表達水平升高且n-6/n-3PUFAs的比值降低。脂肪酸合成基因FASN、ACC、SREBP-1表達與對照相比明顯降低,脂肪酸代謝基因PPARA、PPARG降低。細胞中fat1蛋白表達。(2)獲得了雙酶切和單酶切載體的轉(zhuǎn)基因小鼠。其中轉(zhuǎn)基因雙酶切鼠FO代7只,F1代3只,F2代2只；轉(zhuǎn)基因單酶切鼠F0代7只,F1代28只。轉(zhuǎn)基因雙酶切鼠F0、F1和F2代鼠的肌肉和脂肪組織中n-3 PUFAs含量均升高,n-6/n-3PUFAs比值均降低；轉(zhuǎn)基因單酶切鼠肌肉和脂肪組織中n-3 PUFAs含量降低,n-6/n-3 PUFAs比值升高。(3)雙酶切鼠肌肉組織中脂肪酸相關(guān)基因?qū)崟r定量分析,發(fā)現(xiàn)脂肪酸合成基因FASN、ACC、SREBP-1表達與對照相比明顯降低,脂肪酸代謝基因PPARA、PPARG表達降低。(4)蛋白差異表達和動態(tài)定量分析,發(fā)現(xiàn)單酶切組試驗差異蛋白有99個,其中上調(diào)蛋白62個,下調(diào)蛋白37個；雙酶切組試驗差異蛋白有118個,其中上調(diào)蛋白81個,下調(diào)蛋白37個。(5)利用表達良好的轉(zhuǎn)基因細胞系為核供體生產(chǎn)24枚克隆胚胎,移植受體母畜12頭,有2頭妊娠。其中1頭發(fā)育到期并生下健康犢牛。上述結(jié)果表明,MAR-CAG-Fat-1-PolyA-MAR載體的轉(zhuǎn)基因細胞、轉(zhuǎn)基因小鼠均有良好表達,而且可以生產(chǎn)轉(zhuǎn)基因克隆牛。
[Abstract]:The product encoded by Fat-1 gene is polyunsaturated fatty acid dehydrogenase, which can transform n-6 polyunsaturated fatty acid (PUFas) into n-3 PUFAs. In this study, the MAR matrix attachment regions sequence was used as the starting element to construct the MAR-CAG-Fat-1-PolyA-MAR monocistron expression box, which was transfected into Bovine fetal fibroblasts in Bohai Sea. The monoclonal cells were cultured by limited dilution method, and genomic DNA was extracted for identification. Transgenic monoclonal cell lines were obtained. Then, DNA integration RNAs, protein expression and fatty acid content of transgenic positive cells were identified and analyzed. Transgenic mice were prepared by pMAR-CAG-Fat-1-PolyA-MAR vector (single enzyme digestion vector) and MAR-CAG-Fat-1-PolyA-MAR vector (double enzyme digestion vector). The cloned embryos were prepared by somatic cell nuclear transfer. The results showed that Monoclonal positive cell lines stably transfected with double enzyme digestion vector were obtained. The expression of n-3 PUFAs increased and the ratio of n-6/n-3PUFAs decreased. The expression of fatty acid synthase gene ACCS SREBP-1 was significantly lower than that of control, and the fatty acid metabolism gene PPARAA PPARG was decreased. Transgenic mice with double enzyme digestion and single enzyme digestion vector were obtained. Among them, there were 2 mice in F _ 2 generation and 7 F _ 1 generation in FO generation and 28 F _ 1 generation in F _ 0 generation of transgenic mice. The content of n-3 PUFAs in muscle and adipose tissue of F0 / F 1 and F 2 generation mice was increased and the ratio of n-6 / n-3 PUFAs was decreased. The content of n-3 PUFAs in muscle and adipose tissue of transgenic mice was decreased. The ratio of n-6 / n-3 PUFAs was increased. The results of real-time quantitative analysis of fatty acid related genes in muscle tissue of mice by double enzyme digestion showed that the expression of fatty acid synthase gene FASNN-ACC-SREBP-1 was significantly lower than that of control. The differential expression and dynamic quantitative analysis of fatty acid metabolism gene PPARA-PPARG) showed that there were 99 differentially expressed proteins in single-enzyme digestion group, including 62 up-regulated proteins, 37 down-regulated proteins, and 118 differentially expressed proteins in double-enzyme digested group. Among them, 81 upregulated proteins and 37 down-regulated proteins were used to produce 24 cloned embryos from well-expressed transgenic cell lines. One of the calves matured and gave birth to healthy calves. The results showed that the transgenic cells of MAR-CAG-Fat-1-PolyA-MAR vector were well expressed in transgenic mice and could produce transgenic cloned cattle.
【學位授予單位】：內(nèi)蒙古大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S823

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