Genetic Identification Of Somatic Cell Nuclear Transfer Goats And Genomewide Methylation Profiles In Nuclear Transfer Goats And Cattle

Animal production by somatic cell nuclear transfer (SCNT) offers a range of opportunities in basic and applied research, in agriculture, genetic conservation and human medicine. Microsatellite DNA is dispersed over the whole genome randomly and widely, and microsatellite DNA technology has become the first choice for parentage identification and individual judging for its abundant polymorphisms, codominance and being detected readily by PCR. Therefore, genetic identification of SCNT animals can also be achieved by microsatellite DNA technology. During somatic nuclear transfer cloning, a differentiated cell must become'reprogramming'restoring totipotency in order to undergo development, and DNA methylation is the most important part of epigenetic reprogramming. A great many studies indicated that DNA methylation reprogramming had occurred aberrantly in most preimplantation cloned embryos, and DNA methylation level of postimplantation nuclear transfer fetuses was also significantly different from that of artificial insemination-generated ones, which all might contribute to aberrant phenotype occurance and the low efficiency of cloning. Therefore, analysis of DNA methylation profile on a genome-wide scale in SCNT animals will provide some new insights into the epigenetic mechanisms of occurance of aberrant development in cloned animals, then to contribute to improvement of somatic cell nuclear transfer technology.The present study can be divided into two parts.The first part was performed with two unrelated Lubei White goats to ananlyze 36 microsatellite loci by polymerase chain reaction-polyacrylamide gel electrophoresis (PCR-PAGE) in the begining. Finally, we screened 10 high polymorphic microsatellite loci to be used for genetically identical analysis of the goats cloned from somatic cells. Microsatellite analysis showed that the three cloned goats were genetically identical to their respective donor cells at 9(C1), 10(C2) and 9(F) loci analyzed respectively, whereas different from their recipients at those loci. These results confirmed that the three cloned goats were derived from donor cells.For the second part, the genomewide profiles of DNA methylation in 3 NT goats and 3 NT cattle were analyzed respectively by methylation sensitive amplification polymorphism(MSAP) method for the first time, with non-cloned controls. And totally 38 differential methylation fragments were isolated, corresponding to 38 differential methylation sites, with 28 from cloned goats and 10 from cloned cattle respectively.All fragments were sequenced successfully. According to the results of blasting against bovine genomic sequence database, we found that the 28 differential MSAP fragments from cloned goats exhibited high identity to sequences from bovine genomic sequence database; and 15 of 28 differential MSAP fragments matched sequences of bovine database well (identity over 92%) with near 100% of coverage. Most of differential MSAP fragments showed high homology to the introns of genes predicted by"GNOMON"method. 7 of 10 differential MSAP fragments from cloned cattle also matched bovine database sequences quite well with identity from 97% to 100% and the coverage of differential sequence almost 100%. 4 of 10 differential MSAP fragments showed high homology to relatively uncharacterized bovine genomic sequences or spacer DNA, and the remaining 6 fragments exhibited high homology to introns of genes predicted by"GNOMON"method.According to the results of blasting against genomic seqence database of all species, all differential MSAP fragments showed high identity to interspersed sequences mainly, with over 20bp of coverage and identity from 80% to 100%. Based on the cattle genomic sequence database, 7 of 10 differential MSAP fragments were analyzed by Hpaâ…¡-sensitive PCR method and the remaining 3 fragments by bisulfite sequencing (BSP) method. Finally, 5 differential methylation sites were confirmed with obvious hypomethylation, including C1, C2, C9, C11 and C16. In conclusion, some differential methylation sites were identified in SCNT cattle, the significance of which on the abnormal development of cloned animals need to be further investigated.