| Interspecies cloning may be used as an effective method to conserve highly endangered species and support the development of non-human primate animal models for studying therapeutic cloning and nuclear-cytoplasm interaction. The use of the monkey model for biomedical research can avoid legal, ethical and experimental limitations encountered in a clinical situation. We describe in this study the in vitro development of monkey-pig embryos produced by fusing monkey fibroblasts with enucleated pig oocytes and examine the development of these embryos.1. A Crab-eating Macaque ear marginal tissue fibroblast cell line was successfully established by using a primary explant technique and cell cryoconservation technology. Chromosome analysis showed that the rate of2n is90.16%、86.20%、83.07%passage5,10and30. Cell type was also identified by fluorescent immunocytochemistry and the result indicated that isolated cells were fibroblast cells. cell skeleton was normal assessed by a-tublin staining. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, or mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGF2cl, pDsRedl-N1were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between8.12%and12.35%.The mico-freezing is feasible for cryoconservation of Crab-eating Macaque (Maccaca fascicularis) ear skin fibroblasts.2. Arrest of cells in G0/G1cell cycle phase is desired for somatic cell nuclear transfer procedures. The aim of this study was to determine the cell cycle arresting effects of several medical for different times on different cell cycle stages of Maccaca fascicularis ear-derived fibroblasts. Ear tissue was obtained from a4-year-old male monkey. The tissue pieces were evenly plated in60-mm dish containing2-ml culture medium [DMEM and10%fetal bovine serum (FBS)]. Monolayer cells were passaged in DMEM with10%FBS and harvested with PBS containing0.25%(w/v) trypsin and0.2%(w/v) EDTA when the culture grew to100%confluence. Cells were frozen in DMEM with10%FBS and10%DMSO and stored in liquid nitrogen until used. Cells at the passages three to eight were used for analysis. Cells were analyzed by flow cytometry. The treatments were given after culture with the cells having90-100%confluency. Statistical analyses were carried out by ANOVA using Statistical Package for Social Sciences11.5for Windows (SPSS, Inc., Chicago, IL, USA). Serum starvation increased the percentage of G0/G1phase cells as compared to cycling cells (67.52%)(p<0.05)or cells cultured to confluency. β-mercaptoethanol (10mM), cysteine(2mM) for24hours can improve the rate of GO/G1significantly, the percentage of G0/G1phase cells separately is82.04%and84.66%. The rate of G0/G1was higher than control group when cell was treated by6-DMAP (71.26%) and CB (79.89%) for24hours. The percentage of GO/G1phase cells was79.72%and97.94%when cells were treated by CHX (7.5mg/ml)for24and48hours。The percentage of G0/G1phase cells was76.51%,89.69%and90.49%when cells were treated by CHX (7.5mg/ml)for15μM Roscovitine for24,48and72hours. The cell cycle synchronization in GO/G1phase in the treatment of cells with1.5%and2.0%DMSO for4h (82.86%,86.31%) was significantly higher (p<0.05) than that in the other groups treated for4h (0.5%DMSO for78.84%,1.0%DMSO for78.65%) and the control group (67.52%). Proportion of cells in GO/G1reached better results (p<0.05) for24h with1%(90.45%%),1.5%(91.57%) and2.0%DMSO (98.68%) than0.5%DMSO (78.22%) and the control group (67.52%). In the control group,4.63%of cells underwent apoptosis.1.5%and2%DMSO for4h and1%,1.5%, and2%DMSO for24h caused apoptosis in10.52%,12.75%,10.42%,12.75%,17.07%of cells, respectively. Those results show that, although2%DMSO for24h effectively arrested cells at G0/G1stage, it increased apoptosis of cells significantly (p<0.05). However culture of1.5%and2%DMSO for4h and1%and1.5%DMSO for24h was found to be an appropriate method to obtain more healthy G0/G1cells based on the low percentage apoptotic cells after treatment, which could have a positive impact on somatic cell nuclear transfer in the monkey.3. Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of5-azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in Maccaca fascicularis ear-derived fibroblasts; this may improve NT attributable to the potential reprogramming. Cells were treated with0.08、0.31、1.25、5μM5-azaC for96h;5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by FITC/PI assay. The rate of apoptosis is higher in the group treated (0.08uM:6.91%,0.31μM:8.39%,1.25μM:8.61%,5μM:8.93%) than control group (0μM:7.71%). The normal rate of chromosomal sets of the cell treated with1.25μM5-azaC (76.6%) and5μM5-azaC (68.8%) is lower than control group (87.3%).. Analysis showed no considerable changes in the proportion of cells at the G0/G1-phase of the cell cycle with5-azaC concentrations. The methylation levels in cells with increased concentrations of5-azaC is lower than control group.4. Trichostatin A (TSA), a histone deacetylase inhibitor, could increase histone acetylation, and active gene expression in somatic cells. The present study evaluated the effective dose of TSA, a histone deacetylase (HDAC) inhibitor, for determination of the level of enhancement of histone acetylation in Crab-eating Macaque fibroblasts based on their morphology, growth, apoptosis, cell cycle status and histone acetylation. Ear tissue was obtained from a4-year-old male monkey. The tissue pieces were evenly plated in60mm dish containing2-ml culture medium [DMEM and10%fetal bovine serum (FBS)]. Monolayer cells were passaged in DMEM with10%FBS and harvested with PBS containing0.25%(w/v) trypsin and0.2%(w/v) EDTA when the culture grew to100%confluence. Cells were frozen in DMEM with10%FBS and10%DMSO and stored in liquid nitrogen until used. Cells at the passages three to eight were used for analysis. The number of apoptotic cells were quantified by cytometry after cells were stained with annexin V and PI. The histone acetylation levels were quantified (three replicates) using a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, San Jose,CA, USA) by measuring the level of fluorescence of cells after incubation in histone-specific rabbit primary antibodies and fluorescein isothiocyanate (FITC)-conjugated anti-goat secondary antibodies. TSA inhibited cell proliferation at all tested concentrations in a dose-dependent manner. However, there was slow cell growth for cells treated with0.08and0.1uM TSA compared with those of untreated cells and those treated with other lower concentrations (0.01,0.03and1.0uM).The cells treated with2.0or3.0uM TSA for96h had increased cell sizes and a more flattened and elongated appearance. TSA induced apoptosis in a dose-dependent manner; the rates of apoptosis were3.8±0.12%for0uM TSA,4.0±0.1%for0.01uM TSA,5.1±0.3%0.03uM TSA,6.7±0.5%for0.05uM TSA,9.9±0.5%for0.08uM TSA and12.6±10.7%for0.1uM TSA quantified by cytometry after cells were stained with annexin V and PI. The chromosomal sets of the cell treated with0.01,0.03and0.05uM TSA were normal, whereas a lower proportion of cells treated with0.08and0.1uM were classified as normal. Analysis showed no considerable changes in the proportion of cells at the G1/G0phase of the cell cycle with TSA concentrations. The levels of acetylated histones H4in the cells exposed to various concentrations of TSA for96h were determined quantitatively and qualitatively by FACS and immunocytochemistry, respectively. The nuclei of all the live cells stained uniformly for acetylated histones H4and the levels of these acetylated histones increased in a dose-dependent manner. In conclusion,0.03and0.05uM TSA induced histone hyperacetylation in the Crab-eating Macaque fibroblasts and produced less deleterious effects than other concentrations; these cells might serve as suitable donors for monkey somatic cell nuclear transfer (SCNT).5. We describe in this study the in vitro development of monkey-pig embryos produced by fusing monkey fibroblasts with enucleated pig oocytes and examine the development of these embryos. It shows that monkey-pig cloned embryos can develop to the blastocyst stage when cultured in vitro in PZM-3medium. The effect of IVM medium of TCM-199and NCSU-23on the rate of MII and cumulus expansion is similar. The percent of enucleated pig oocytes is higher at IVM44h,48h than60h. PZM-3embryo culture medium is better than NCSU-23. The higher death rate was got by electrical activation1.4k V/cm,80μs,1DC than otOher two groups (1.2k V/cm,30μs,2DC;2.0k V/cm,50μs,1DC). The rate of blastocyst development was20.45%,7.35%,5.95%,8.67%when the embryo was reconstructed by pig-Fetal fibroblast, pig-cumulus, pig-ear, and monkey-ear cell. The effect of different passage cell at1-3,4-6,7-10on embryo development had no difference. The rate of blastocyst development was10.7%,8.7%and8.0%when the embryo reconstructed was cultured in PZM-3, HECM-9and mixed PZM-3and HECM-9. The rate of blastocyst development was1.0%,0.3%and1.3%when the embryo reconstructed was cultured in37℃,39℃(4D)+37℃,37℃(4D)+39℃condition. The effect of different co-culture with different cell on embryo development did not improve the development of embryo. The development of embryo was increased when the cell was treated with15μM Roscovitine for48hours in both SCNT(14.23%) and iSCNT (15.05%). If cell was treated with0.03μM TSA or0.31μM5-azaC for96hours, the rate of blastocyst development was7.63%(control),8.54%,7.42%。6. In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, mtDNA of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos (NT-embryos) where nuclear donor and oocyte are from two distantly related species, the distribution of mtDNA species is not known. Here we determined levels of monkey and pig mtDNAs in monkey embryos reprogrammed by pig oocytes (monkey-pig embryos). Quantification using a real-time PCR method showed that both monkey and pig mtDNAs co-exist in NT-embryos of preimplantation stages, with maternal mtDNA as the dominant. Single NT-embryo at1-cell stage after fusion contain3.01×104copyies of monkey mt DNAs and6.78×106copyies of pig mtDNAs. The rate of monkey mtDNAs per total mtDNAs is0.44%,0.82%,0.92%,1.19%,1.47%at1-cell,2-cell,4-cell,8-cell,and blastocyst stage.The distribution of mtDNA is not balanced followed the development of iSCNT embryos. |
PDF Full Text Request