Several mammalian species have been successfully cloned by somatic cell nuclear transfer (NT), but only a small proportion of the embryos produced using adult or fetal somatic cells develop into living young (typically between 0 and 4%). Clearly, the causes of low efficiency must be identified if the medical and agricultural potentials of cloning procedures are to be realized. In cloning mammals by somatic cell nuclear transfer (NT), the doner nucleus requires epigenetic reprogramming to a totipotent ground state. The incomplete reprogramming of donor somatic cell nuclei leading to aberrant (or even lack of) expression of some developmentally important genes has been implicated as a primary reason for this low efficiency.
Some genes important for early embryogenesis have been studied in cloned preimplantation embryos. Examination of the expression of many imprinted genes in cloned mice that survived to birth has shown that some of them are abnormal. Many cloned offspring die shortly after birth and often exhibit phenotypic abnormalities, but the reasons for these abnormalities have not been clarified. One objective of the present study was to investigate the expression of developmentally important genes in the organs of cloned bovine of neonatal death to determine the possible genetic causes of the neonate’s death and of the organ abnormalities in animals produced using NT techniques.
The X-inactive specific transcript (Xist) plays a crucial role during X-chromosome inactivation (XCI). Hsp70.1 (70-kDa heat-shock protein) is a stress protein involved in facilitating protein folding and assembly as well as in stabilizing damaged proteins for repair and degradation in cells. PCAF (p300/CFBP-associated factor), which has histone acetylase activity, is involved in cell-cycle arrest, transcriptional activation, plays a role in transcriptional activation, cell-cycle arrest, and cell differentiation in cultured cells. Vascular endothelial growth factor (VEGF), platelet-derived growth factor receptor a (PDGFRa), bone morphogenetic protein 4 (BMP4), fibroblast growth factor receptor 2 (FGFR2), and fibroblast growth factor 10 (FGF10) play important roles in embryo development and organogenesis. In the present study, the transcripts of Xist, FGFR2, PDGFRa, Hsp70.1, VEGF, BMP4, and FGF10 were examined in tissues from both deceased neonatal clones and normal control calves.
Cloning is a multistep procedure, and many factors may affect cloning efficiency. The donor cell type, age, and cell-cycle stage are factors generally thought to affect cloning efficiency in mammals. In somatic cloning of cattle, many kinds of differentiated cells have been tested, but most investigators have used skin fibroblasts as donor nuclear material. Fibroblasts offer several advantages for future applications of somatic cloning. First, a skin biopsy specimen can be easily obtained from a valuable animal, so the production of large numbers of genetically identical copies of the animal are possible. Second, fibroblasts can be easily cultured and stored frozen, so they probably are good candidates for genetic modification to produce transgenic animals. Age of the fibroblast donor cell has no effect on in vitro development of bovine NT embryos, but other reports indicate that clones derived from adult cells frequently abort during the later stages of pregnancy and that calves developing to term show a higher number of abnormalities than those derived from newborn or fetal cells. In the present study, nine deceased cloned bovines were derived from adult fibroblast (AF) and fetal fibroblast (FF). The other objective of the present study was to examine the effect of donor cell age on the expression of developmentally important genes in cloned bovines.
Tags: developmental biology, early development, gene regulation, growth factors, stress