Identification Of Nuclear Transfer Goats And Analysis Of H19 Gene Regulation And Expression

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.