| Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic cell nuclear transfer (NT) requires epigenetic reprogramming of the differentiated state of donor cell to a totipotent, embryonic ground state. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. Examination of gene expression in cloned animals has largely been limited to preimplantation embryos for a small number of genes important for early embryogenesis. Cloning by somatic nuclear transfer is an inefficient, process in which some of the cloned animals die shortly after birth and display organ abnormalities. In somatic cloning of cattle, fibroblast was often used as donor nuclear. Fibroblasts offer several advantages for future applications of somatic cloning. First, the skin biopsy can be easily gained from a valuable animal, so that to produce large numbers of genetically identical copies of the animal are possible. Secondly, fibroblasts can easily cultured and stored frozen, and which are probably good candidates for genetic modification to produce transgenic animals.In this research, the donor nuclei were obtained from skin fibroblast cells of a female four-year-old elite Holstein cow and from fetal fibroblast cells of a 40-day-old female fetus. About 27.9% (n=283) and 37.9% (n=294) of reconstructed embryos derived from adult fibroblast cell and from fetal fibroblast respectively developed into blastocysts, with 11(14.9%, n=74) and 10 (22.7%, n=44) of the transferred embryos developing into full-term calves. Six of the eleven calves from adult fibroblast cell survived and remained healthy, and five died. Six often calves from the fetal fibroblast cells survived and remained healthy, while four died. All of the deceased cloned cattle died within 48 hours of birth. The abnormalities were observed at necropsy and tissue pathologic section of deceased cloned cattle' organs, including heart, liver, spleen, lung, kidney and brain, and in which the abnormalities of heart and lung were serious.In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we have examined expression patterns of 22 developmentally important genes in six organs (heart, liver, spleen,lung,kidney and brain )of both neonatal death cloned bovines and normal control calves produced by artificial insemination (AI) using real-time quantitative RT-PCR. The genes include four genes of modifying chromatin strcture (DNNT, PCAF, MeCP2, EED) eight genes of insulin-like growth factors systems (IGF1, 1GF2, IGF1R, IGF2R, IGFBP1, IGFBP2, IBGBP3 and IGFBP4)s fibroblast growth factor (FGF2 and FGF10) and fibroblast growth factor receptor (FGFR1 FGFR2 )and other six developmentally important genes( EGFR, PDGFRa, VEGF, BMP4, Hsp70.1 andAberrant expressions of sixty genes were found in these clones The aberrant expressions of IGFBP4 were observed in five organs; IGF2R and BMP4 in four organs; five genes of DNMT, PCAF, IGF2, VEGF Hsp70.1 in three organs; three genes of FGF10 FGFR1 and PDGFRa in two organs, and aberrant expressions of five genes ofIGFlR IGFBP2 IGFBP3 EGFR and Xist were only observed in one organs.For the studied genes, spleen was the organ that less affected by gene dysregulaion in which four genes were aberration, whereas heart was the most affected one, in which ten genes were aberration, in liver, seven genes were aberration and five genes were up-regulation. In lung, kidney and brain, six genes were aberration and kidney was the only organ with all dysregulated genesof down-regulationComparing the gene expression between the two types clones with each other and eight gene (IGF2R, Hsp70.1 Xist PDGFRa BMP4 IGFBP4 FGF10 and IGF2) expression were found having significant difference between AF cell-derived and FF cell-derived clones.For the sixty aberrant e... |
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