【作者】 李长雷；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2010， 硕士

【摘要】 动物核移植技术产生之后,有力的促进了生命科学领域的发展,但在动物出生后,采用何种方法证明动物是由无性繁殖而来,这是动物核移植研究中一个十分关键问题;而且通过核移植技术得到健康动物的效率非常低,核移植动物的流产率和新生儿死亡率远高于体外受精和人工受精得到的动物,即使是存活下来的核移植动物也常伴有表型异常和不同程度的发育缺陷,研究表明表观重编程异常是核移植胚胎发育异常的重要原因之一,而表观重编程异常与基因组印记有很大关系。DNA甲基化是基因印记发生和维持的主要机制。印记基因的分子机理与印记基因中胞嘧啶甲基化尤其是CpG岛的甲基化密切相关。本实验通过微卫星技术鉴定转基因核移植山羊和核供体细胞的基因型是否完全一致。运用亚硫酸盐法测定并比较了H19基因CpG岛在转基因核移植山羊和同期成活普通山羊(对照组)不同组织中的甲基化率,检测在转基因核移植山羊部分组织中H19基因CpG岛是否发生了异常甲基化。采用实时荧光相对定量方法测定出转基因核移植山羊和对照组山羊不同组织中H19基因mRNA的相对表达量。验证转基因核移植山羊H19基因CpG岛甲基化模式发生异常是否会引起其mRNA相对表达量的异常,判定H19基因CpG岛甲基化结果和相对量是否对应。进一步揭示山羊H19基因的功能和作用,为提高核移植效率提供一定的技术支持。实验结果表明:1、利用五对多态性引物:SR-CRSP1, SR-CRSP5, SR-CRSP6, SR-CRSP7,SR-CRSP24对转基因核移植山羊,供体细胞,代孕母羊和对照组母羊进行基因型判定,微卫星DNA多态性分析结果表明,转基因核移植山羊和核供体细胞的基因型完全一致,这说明两者在这些位点上具有完全相同的遗传信息。因此,判定本实验中获得的转基因核移植小羊来源于核供体细胞,转基因核移植山羊生产获得成功。2、运用亚硫酸盐法分别检测了死亡转基因核移植山羊(2号小羊)和同期普通山羊(3号小羊,对照组)肝脏、胎盘(包括存活转基因核移植山羊,1号小羊)、肾脏、肺脏和心脏组织中H19基因CpG岛甲基化水平。结果表明,胎盘组织中,2号小羊H19基因第5个CpG岛的甲基化水平显著高于对照组(70% Vs 49.41%,P<0.05);肺脏组织甲基化水平显著低于对照组(63.53% Vs 88.24%,P<0.05);其他各组差异不显著(P>0.05)。同时2号小羊胎盘组织中H19基因第5个CpG岛的甲基化水平显著高于1号小羊(70% Vs 51.18%,P<0.05),但1号小羊胎盘组织甲基化率与对照组3号小羊相比较差异不显著(51.18% Vs 49.41%,P>0.05)。以上结果说明,H19基因在死亡转基因核移植山羊部分组织中DNA甲基化重编程异常。3、运用实时定量PCR法测定出H19基因在2号小羊和3号小羊肝脏、胎盘(包括1号小羊)、肾脏、肺脏和心脏中的相对表达量。显示2号小羊和3号小羊在肝脏、肾脏和心脏组织中H19基因的相对表达量差异不显著(P>0.05),在胎盘组织中的相对表达量显著低于对照组和1号小羊(883.3 Vs 1264.5,883.3 Vs 1197.6,P<0.05),在肺脏组织中的相对表达量显著高于对照组(1003.4 Vs 515.5,P<0.05);但是2号和3号小羊H19mRNA在胎盘组织中的相对表达量差异不显著(1197.6 Vs 1264.5,P>0.05)。说明H19基因第5个CpG岛甲基化异常影响H19基因在上述五类组织中的mRNA正常表达。这也可能是导致转基因核移植动物死亡的重要因素之一。更多还原

【Abstract】 After the animal nuclear transfer technology be invented which effective promotion of the development of life sciences, however, the animal was born later, put to use any way to proved animal from the donor cell, which is a very obtain a healthy animals by nuclear transfer, the abortion rate and neonatal mortality rates of animals by nuclear transfer are far higher than that in vitro fertilization and artificial insemination of animals, even surviving nuclear transfer animals are often accompany epigenetic variation and varying degrees of developmental defects, the research indicated that epigenetic reprogramming abnormal is one of the important reasons in nuclear transfer embryos dysplasia, and the epigenetic reprogramming abnormal was caused which relation with the genomic imprinting. DNA methylation is the main mechanism of the occurrence and maintenance of gene imprinting. The molecular mechanism of imprinting gene closely related to cytosine methylation of imprinted genes, especially the CpG islands.In this study, to identification the genotype of transgene nuclear transfer goat and nuclear donor cells whether are exactly same through the micro-satellite technology. To investigate and compare the H19 gene CpG island methylation profiles in different tissues of transgene nuclear transfer goat and age-matched common goat fetus(control group) by using bisulfite sequencing. Detection the H19 gene CpG island of in some tissues of transgene nuclear transfer goat whether abnormal methylation occurred. To investigate and compare the H19 gene mRNA relative expression in different tissues of transgene nuclear transfer goat and age-matched common goat fetus(control group) by using real-time PCR, verify H19 gene CpG island methylation patterns of transgene nuclear transfer goat whether can lead to its mRNA relative expression abnormal,determine the results of H19 gene CpG island methylation whether corresponding with the relative expression. Further reveal the function of H19 gene in goat and provide some technical support.to improve the efficiency of nuclear transfer .Experiments results indicate:1. Using five pairs of polymorphic primers SR-CRSP1, SR-CRSP5, SR-CRSP6, SR-CRSP7and SR-CRSP24 to detection the genotype of transgene nuclear transfer goat, donor cell, surrogacy goat and goat of control group, the polymorphic results of micro-satellite DNA showed that transgene nuclear transfer goat and nuclear donor cells have the same genotype, which indicating that locis have identical genetic information. So, determined the transgene nuclear transfer goat origin from the nuclear donor cells in this experiment, Success the experiment of transgene nuclear transfer goat product!2. We detected the H19 gene CpG island methylation of liver, placenta(including the survival transgene nuclear transfer goat, number 1), kidney, lung and heart in the dead transgene nuclear transfer goat(number 2)and the age-matched normal goat fetus (number 3, control group) by using bisulfite sequencing. Results indicated that methylation levels of the fifth CpG island of H19 gene in goat of number 2 was significant high compared with that in the control in placenta(70% Vs49.41, P<0.05 ).Reversely, the methylation levels was significant low compared with that in the control in lung(63.53% Vs 88.24%,P<0.05), The differences of others groups were insignificant(P>0.05). And methylation levels of the fifth CpG island of H19 gene in goat of number 2 was significant high compared with that in goat of number 1 in placenta (70% Vs 51.18%,P<0.05), however, methylation levels of the fifth CpG island of H19 gene in goat of number 1 was insignificant compared with that in goat of number 1 in placenta(51.18% Vs 49.41, P>0.05). Results showed the abnormal DNA methylation reprogramme of H19 gene occurred in some tissues of dead transgene nuclear transfer goat.3. Detected the H19 gene mRNA relative expression of liver, placenta(including goat of number 1), kidney, lung and heart in goat of number 2 and goat of number 3 by real time PCR. Results indicated that relative expression levels of H19 gene in goat of number 2 was insignificant compared with that in the goat of number 3 in liver, kidney and heart(P>0.05), and in goat of number 2 was significant low compared with that in the control and in goat of number 1 in placenta (883.3 Vs 1264.5,883.3 Vs 1197.6,P<0.05), in goat of number 2 was significant high compared with that in the control in lung(1003.4 Vs 515.5,P<0.05);but relative expression levels of H19 gene in goat of number 2 was insignificant compared with that in the goat of number 3 in placenta (1197.6 Vs 1264.5,P>0.05). Results showed the abnormal DNA methylation proflies of H19 gene occurred in some tissues, which affected normal expression levels of H19 gene, indicating that aberrant DNA methylation reprogramme may be one of the important factors for the death of transgene nuclear transfer animals.更多还原