【作者】 张东；

【导师】 周欢敏；

【作者基本信息】 内蒙古农业大学 ， 动物发育生物学与生物技术， 2011， 博士

【摘要】 fat-1基因编码ω-3 PUFAs脱氢酶,可以将PUFAs从ω-6转化为ω-3形式。本研究目的是通过动物转基因技术将fat-1转入绵羊的基因组,再利用动物克隆技术创造能产生ω-3 PUFAs的转基因绵羊,从而培育含ω-3 PUFAs丰富的转基因肉羊新材料。以我区绵羊为对象,摸索了绵羊克隆胚的构建和胚胎移植体系;建立雄性绵羊胎儿的皮肤成纤维细胞系;克隆了秀丽隐杆线虫的fat-1基因,构建目的基因表达载体;再将其转染到绵羊成纤维细胞基因组中,经G418筛选,PCR鉴定后选择阳性细胞建立转基因细胞系;通过体细胞克隆动物技术构建并生产转基因克隆胚并移植,待移植后代出生后鉴定并检测其肌肉中ω-6和ω-3 PUFAs的相对含量及比值;以获得转基因的具有保健功能的肉羊新品种。1.绵羊体细胞克隆体系的优化本实验主要是对体细胞克隆胚移植胚龄的摸索。采用组织块种植法分离培养了三种雌性成年绵羊耳皮肤成纤维细胞系,分三个阶段进行绵羊体细胞克隆体系的优化。第一阶段构建了来源于1号细胞的重构胚272枚,体外发育到桑椹胚期移植入17只受体羊中,有一只妊娠,妊娠率为5.88%,产羔羊1只,经鉴定为克隆羊。第二阶段构建了来源于2号细胞的重构胚683枚,分别在胚胎发育的不同时期进行了胚胎移植,共移植了35只受体羊。结果显示,在3只移入的胚龄是1-细胞期胚胎的羊中,有一只妊娠并产羔1只,经鉴定为克隆羊。第三阶段构建了来源于3号细胞的重构胚197枚,在1-细胞期阶段全部移入了11只受体羊中,结果有两只羊妊娠,妊娠率为18.18%,生产了3只克隆羊,产羔率为27.27%。有效地的重复了第二阶段实验。待克隆羔羊性成熟和体成熟后对其进行了自然交配,成年体细胞克隆羊正常顺产2胎,均是双胞胎。表明获得的克隆羊具有正常的生殖和哺育后代的能力,进一步证明,本实验室建立了相对稳定的的体细胞克隆绵羊技术体系。2.转基因体细胞克隆体系的建立采用组织块种植法分离纯化了约40日龄绵羊胎儿成纤维细胞;克隆了秀丽隐杆线虫fat-1基因,同时对其进行了密码子的优化与合成,构建了pTFK-c和pTFK-o两个真核表达载体。pTFK-o转染绵羊胎儿成纤维细胞,经G418压力筛选后进行转基因细胞的鉴定。结果表明获得的7个细胞克隆中有5个细胞系的基因组中整合有目的基因,对获得的转基因细胞进行染色体数目分析,其核型正常率为69.77% (2n=54),符合用于转基因克隆的要求。以筛选到的7号转fat-1基因绵羊胎儿成纤维细胞系为核供体,绵羊去核卵母细胞胞质为核受体生产转基因克隆胚。采用抽吸法共回收卵母细胞4881枚,成熟培养18h成熟率为72.87%。实验共进行去核操作绵羊卵母细胞3557枚,用于融合的卵数是3305枚,共融合3040枚,融合率为91.39%。共分三组移植了处于1-细胞期的2909枚胚胎到230只羊体内,1组移植了146只受体羊,妊娠5只,妊娠率为3.42%,产羔5只,产羔率为3.42%;2组移植了60只受体羊,妊娠18只,妊娠率为30%,产羔19只,产羔率为31.67%;3组移植了24只羊,妊娠5只,妊娠率为20.83%,产羔6只,产羔率为25%。后2组实验结果进一步优化了体细胞克隆技术体系。3.转fat-1基因克隆绵羊的出生与鉴定通过实验共产羔30只,其中19只存活。对获得的30只中的29只转基因克隆羔羊进行基因组水平检测,结果显示有27只羔羊基因组中含有目的基因。克隆羊肌肉组织中ω-6和ω-3PUFAs的相对含量检测结果显示,在检测的23只羊中有18只羊都表达了有活性的脂肪酸脱氢酶,其ω-6/ω-3 PUFAs比值都有不同程度的降低,平均值为16.45,其ω-3 PUFAs的平均值为0.194,和对照公羊平均值0.147相比提高了31.97%,有效的改善了绵羊体内ω-6和ω-3 PUFAs的相对比例和含量。更多还原

【Abstract】 Fat-1 gene encodingω-3 polyunsaturated fatty acids (PUFAs) desaturase, can convert polyunsaturated fatty acids fromω-6 toω-3 form. The aim of study present here was to obtain transgenic sheeps, which contained abundantω-3 PUFAs in their muscles, through transferring the fat-1 gene into ovine genome and then production the transgenic cloned sheep by animal cloning technology. By means of our predominant sheep species, we optimized the construction and embryo transferring system of ovine cloned embryo, established male ovine fetal fibroblast cell line, cloned the fat-1 gene from Caenorhabditis elegans and constructed the expression vector containing purpose gene. In vitro cultured ovine fetal fibroblast cells were transfected with this vector. The stable transfected cell lines were established through G418 screening, and the transgenic cells were identified by PCR. Using the nuclei from selected donor cells, ovine embryos were produced via nuclear transfer technology and the reconstructed embryos were transferred to recipients via embryo transfer technology. Transgenic generations to be identified and detected the relative amount and ratio ofω-6 andω-3 PUFAs in the muscle after the birth, so that to obtain transgenic sheep with new varieties of health functions.1. Optimization of sheep somatic cell cloning systemThe aim of this experiment is to optimize transferring system of somatic cell cloned embryos on developmental stages of reconstruction embryo. Three female ovine ear skin fibroblast cell lines were isolated and cultured by attachment of tissue pieces and the exploration divided into three stages.The first stage, we constructed 272 cloned embryo from No. 1 cell line, than transferred into 17 recipient female sheeps when the embryos developed in the morula stage in vitro. There is one pregnancy and the pregnancy rate was 5.88%. One lamb was born, and it was identified as Cloned sheep.The second phase we constructed 683 cloned embryo from No. 2 cell line, than transferred into 35 recipients when the embryos developed in different stages in vitro. The results showed that, there was a pregnancy in 3 recipients which were transferred the 1-cell embryos, and born one lamb which was identified as Cloned sheep.The third phase we constructed 197 cloned embryo from No. 3 cell line, than transferred into 11 recipients when the embryos developed in 1-cell stage in vitro. The results showed that, there were two pregnancy and the pregnancy rate was 18.18%. Three lambs were born and the lambing rate was 27.27%. This result effectively repeated the second phase of experiments. The cloned sheep was wildly paired with ram after it developed to sexual and physical maturity. The adult somatic cell cloned sheep delivered two twins. The results showed that we obtained cloned sheep with normal reproductive and feeding ability for offsprings , and further prove that the laboratory had established a relatively stable sheep somatic cell cloning technology system.2. Establishment of transgenic somatic cell cloning systemThe male ovine fetal fibroblast cells were isolated and cultured by attachment of tissue pieces from 40d old ovine fetal. We cloned the fat-1 gene from Caenorhabditis elegans, optimized the fat-1 gene sequence according to ovine hobby codons, and synthesis the optimized fat-1 gene sequence. Then we constructed pTFK-c and pTFK-o eukaryotic expression vectors. Sheep fetal fibroblasts were transfected by pTFK-o, screened by G418, and identified by PCR. The results showed that 5 cell lines genome had integrated the target gene in 7 clones obtained. The chromosome number analysis of the transgenic cell showed that the normal karyotype was 69.77% (2n = 54), so the transgenic ovine fetal fibroblast cells gave a good fit to transgenic cloning manipulation. The transgenic ovine fetal fibroblast cell line 7 as nuclear donor cell and enucleated oocytes served as recipient cytoplasm to produce nuclear transferred embryos. Totally 4881 oocytes were collected by aspiration, 72.87% of them were matured after 18 hours culture and 3557 oocytes were enucleated and 3305 enucleated oocytes were treated for electrofusion with donor cells. Among them, 3040(58.4%) couplets were fused and the fusion rate was 91.39%. Totally 2909 reconstructed 1-cell embryos divided into three groups were transferred into 230 recipients. Group 1 transferred into 146 recipients, and there were 5 pregnant sheeps and pregnancy rate was 3.42%, lambing 5 and lambing rate was 3.42%. Group 2 transferred into 60 recipients, 18 pregnancy and the pregnancy rate was 30%, 19 lambing and lambing rate was 31.67%. Group 3 transferred into 24 sheeps, 5 pregnancy and pregnancy rate was 20.83%, 6 lambing and lambing rate of was 25%. The results of last 2 groups further optimized the somatic cell cloning technology system.3. Birth and identification of fat-1 transferred cloned sheepsWe obtained 30 lambs and 19 survived. There were 27 ovine genome contains fat-1 gene in 29 lambs. The detection results of relative amount ofω-6 andω-3PUFAs in cloned sheep muscle tissue showed that, there were 18 in the 23 sheeps expressed fatty acid desaturase, theω-6/ω-3 PUFAs ratios of them were lower, and the average was 16.45. The average of relative amount aboutω-3 PUFAs was 0.194, which increased 31.97% compared to that of control rams (0.147). This work effective improved theω-6 andω-3 PUFAs relative proportion and content.更多还原