Morphology, Expression And DNA Methylation Status Of Imprinted Genes In Tissues Of Transgenic Cloned Calves

Somatic cell nuclear transfer(SCNT) is an important bio-engineering technology which has broad application potential and practical value in animal production, animal breeding, disease resistance, medical research and protection of endangered species, etc. However, the efficiency of the nuclear transfer is still very low at present. Pregnancy miscarriage and low survival rate usually appear. Survival animals are usually accompanied by various degrees of developmental defects and abnormal phenotypes, and associated with abnormal development of the placenta. Studies have shown one of the major causes of abnormalities in nuclear-transfer animal is the abnormality in gene reconstructed programming in the process of nuclear recombination, which appears as changes of genome epigenetic modification and abnormalities in gene expression of cloned animals.In order to verify the existence of morphological abnormalities in cloned cattles, detect the expression and epigenetic modification of nuclear genes in tissues, and determine whether they are related to abnormal phenotypes of cloned cattle, we chose stillborn and liveborn cloned calves compared with normal ones, and used three important develop-regulating imprinted genes as parameters. The histomorphologic features were observed and the gene expression and DNA methylation status of cloned calves were detected.In this test, paraffin, HE staining, real-time quantitative (Real-time PCR), bisulfite sequencing (BSP) and the combined bisulfite restriction enzyme analysis (COBRA) were used to study the morphological changes of transgenic cloned stillborn calves, and detect the mRNA relative expression and DNA methylation status in DMR of three imprinted genes (H19, IGF2R, XIST) in muscle, liver, spleen, lung, kidney tissues of both stillborn and liveborn cloned calves.1. Staining slices of liver, spleen, lung and kidney of stillborn calves were observed under light microscope and showed no significant abnormality, so the overall organ morphology was normal.2. In muscle, liver, spleen, lung and kidney tissues of transgenic cloned calves, mRNA expression levels of H19, IGF2R and XIST in both stillborn and liveborn groups were significantly different from those in normal group. 3. In muscle, liver, spleen, lung and kidney tissues of transgenic cloned calves, DNA methylation level of DMRs of H19 and XIST, ICR of IGF2R in both stillborn and liveborn groups were significantly different compared with normal group.4. Abnormal mRNA expression and DNA methylation levels of three genes in five tissues were more severe in stillborn group than in liveborn ones.