56CARDIOMYOCYTES FOR THE STUDY OF DEDIFFERENTIATION IN BOVINE NUCLEAR TRANSFER
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Nuclear transfer facilitates the study of the dedifferentiation process of differentiated somatic cells. Cardiomyocytes are a good model of terminally differentiated cells showing a unique gene expression pattern of cardiac marker genes. The purpose of this study was to test bovine cardiomyocytes as donor cells in nuclear transfer. Cardiomyocytes were isolated from fetal heart muscle (3–5 months of gestation), which were obtained at the abbatoir and immediately perfused with cold Custodiol (Dr. Franz Köhler Chemie, Germany) to reduce metabolism and protect the cells against ischaemia. Subsequently, hearts were perfused with collagenase in Krebs-Henseleit buffer (KHB) to dissociate the tissue and isolate single elongated, contractile cells. For nuclear transfer and fusion the cardiomyocytes were rounded up by exposure to increasing calcium concentrations (2.5–200μM) in the culture medium before the cells were incubated in suspension for 46–48 hours in MEM medium plus 10% FCS. Nuclear transfer was performed as described earlier (Lucas-Hahn et al., 2002, Theriogenology 57, 433). As a control, adult female fibroblasts were employed. Fusion rate, cleavage (day 3 of in vitro culture) and development up to the morula/blastocyst (day 7 of in vitro culture) were recorded and statistically analysed with Student’s t-test. A total of 243 nuclear transfer complexes with cardiomyocytes and 127 with fibroblasts were produced. Fusion rates for cardiomyocyte complexes were significantly (P<0.001) lower (28.8%) compared to fibroblasts (84.3%). Cleavage rates were 48.1% for cardiomyocytes and 62.8% for the fibroblast-derived embryos. The developmental capacity to the morula/blastocyst was significantly (P<0.01) reduced for cardiomyocyte (9.4%) compared with the fibroblast-derived (32.4%) reconstructed embryos. Most of the Day 7 embryos were frozen for investigation of gene expression patterns of cardiac marker genes. Staining with Hoechst 33342 for counting total cell numbers revealed that 87.3±20.9 blastocysts were derived from fibroblasts and 100 blastocysts from cardiomyocytes. These results indicate that nuclear transfer with terminally differentiated cardiomyocytes is possible, although with reduced rates. Studies are underway to analyze the gene expression of cardiac marker genes in reconstructed embryos to gain insight into dedifferentiation after nuclear transfer using cardiomyocytes as a model. This study was supported by Deutsche Forschungsgemeinschaft (DFG; Ni 256/16-1)We examined the effects of treatment with histone deacetylase inhibitors (HDACi), trichostatin A (TSA) and scriptaid (SCR), on the blastocyst formation rate in bovine somatic cell nuclear transferred (SCNT) embryos derived from fibroblast cells. Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors to produce SCNT embryos (L1, L2 and L3 embryos, respectively). In Experiment 1, we compared the in vitro developmental competence of L1 embryos treated with various concentrations of TSA for different time periods following chemical activation. Embryos treated with 5 nM TSA for 20 h showed a significantly increased blastocyst formation rate compared with untreated controls. In Experiment 2, we examined the effect of TSA (5 nM) treatment of L1, L2 and L3 embryos as well as the effect of treatment of L1, L2 and L3 embryos with various concentrations of SCR on in vitro developmental competence. It was found that 5 nM TSA treatment significantly increased the blastocyst formation rate in L1 and L3 embryos but did not have an influence on the development of L2 embryos. On the other hand, 5 nM SCR treatment significantly increased the blastocyst formation rates of L1 and L2 embryos compared with controls. However, there was no significant increase in the blastocyst formation rate of L3 embryos when they were treated with SCR. In Experiment 3, acetylation of H4K12 was examined in donor cells and pronuclear-stage L1, L2 and L3 embryos treated with 5 nM TSA or 5 nM SCR by immunostaining. The level of H4K12 acetylation was different among donor cells. The staining intensities in the TSA-treated L1 and L3 embryos and SCR-treated L2 embryos were significantly higher than those of untreated embryos. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, the optimal treatment conditions may differ among donor cell lines.
Trichostatin A
Histone deacetylase inhibitor
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Interstitial collagenase
MMP1
c-Fos
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The present study evaluated the effect of Scriptaid, a novel histone deacetylase inhibitor (HDACi), on the in vitro development of somatic cell nuclear transfer (SCNT) bovine embryos. Average fluorescence intensity of two epigenetic markers (H3K9ac and H3K9m2) at two-cell, eight-cell, and blastocyst stages, and the expression levels of two developmental important genes (Oct4 and IFN-t) at the blastocyst stage were also examined to assess the influence of Scriptaid on the nuclear reprogramming of bovine SCNT embryos. The results showed that treatment with 500 nM Scriptaid for 14 h after activation significantly increased the cleavage rate, blastocyst formation rate, and blastocyst hatching rate of SCNT embryos compared with those of nontreated counterparts, but the total number of blastomeres per blastocyst did not differ. Scriptaid treatment also significantly increased the immunofluorescent signal for H3K9ac in SCNT embryos at two-cell, eight-cell, and blastocyst stages, and the fluorescent signal for H3K9m2 was decreased at two-cell, eight-cell, and blastocyst stages. The expression levels of Oct4 and IFN-t were significantly higher in Scriptaid-treated SCNT blastocysts than in Scriptaid nontreated SCNT blastocysts. The results indicated that Scriptaid treatment improved the in vitro developmental capacity and the nuclear reprogramming of bovine SCNT embryos.
Reprogramming
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Summary Cloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro . Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.
In vitro maturation
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The effects of caffeine and scriptaid(histone deacetylase inhibitors,HDACi) on the blastocyst formation rate and blastocyst cell numbers in sheep somatic cell nuclear transferred(SCNT) embryos derived from cumulus cell were examined.Results showed that the blastocyst cell numbers were significantly increased by addition 2.5 mmol/L or 5 mmol/L caffeine after fusion.The blastocyst rate of SCNT embryos was the highest(24.31%)when 0.2 μmol/L scriptaid was added to the embryo culture medium,which was significantly higher than that of control group and 0.8μmol/L scriptaid addition group(P0.05).Interestingly,the blastocyst cell number was the highest in 0.2 μmol/L scriptaid addition group too.In conclusion,the blastocyst cell number can be improved significantly by addition caffine though the rate of blastocyst of cloned embryos was not increased significantly;the blastocyst developmental rate of SCNT embryo can be improved notably by addition scriptaid.
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si e s------------------------ABSTRACT------------------------- P4 initiates specifically the degradation of interstitial collagen types I-III. This enzyme is thus directly involved in the remodeling of the connective matrix. Fibroblasts are considered as the major source of interstitial collagenase in many tissues. However previous studies shown that liver fibroblasts did not spontaneously produce the enzyme. We have thus measured the steady-state levels of mRNA for interstitial procollagenase and quantified interstitial collagenase activity in cultured human liver fibroblasts, in presence or not of interleukin-1 or tumor necrosis factor. We demonstrate that human liver fibroblasts have the capacity for producing interstitial collagenase and that this production is regulated at a transcriptional step. We suggest that the liver fibroblast could represent a key cell for therapeutic strategies of fibrosis reversal.
Interstitial collagenase
Interstitial cell
Hepatic stellate cell
Microbial collagenase
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Ovine sperm extract(OSE) was used to activate ovine somatic cell nuclear transfer(SCNT)embryos and goat-ovine interspecies somatic cell nuclear transfer(ISCNT) embryos.The effects of different dose of OSE activation on the development capacity of the reconstructed embryos were compared according to the cleavage rate,blastocyst rate and blastocyst cell number.The results showed that OSE could effectively activate the SCNT and the ISCNT embryos and the optium concentration of OSE was 5 mg/mL,the activation effect was significantly(P0.05) better than other groups.The effects of OSE activation or chemical activation(using ionmicin and 6-DMAP) on the development capacity of the reconstructed embryos were evaluated,the results indicated that there was no significant difference in blastocyst rate and blastocyst cell number when the ISCNT embryos were activated by using the OSE or the chemical activation method and there was no significant difference in blastocyst rate when the SCNT embryos were activated,however,there was a significant increase(P0.05) in blastocyst cell number when the SCNT embryos was activated by using OSE compared with the chemical activation method(72.4 vs 65.2,P0.05).
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Human skin
Microbial collagenase
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The purpose of this study was to develop an improved zona-free method of goat somatic cell nuclear transfer (SCNT) that has both ease of operation and efficiency. The main steps involved were: (1) optimization of in vitro oocyte maturation, (2) parthenogenetic activation of zona-free oocytes, (3) SCNT of zona-free anaphase II-telophase II (AII-TII) oocytes that subverted the need for long term UV-exposure of the oocytes, and (4) in vitro culture of groups of cloned embryos in wells in a highly efficient continuous serum-free embryo medium to the blastocyst stage before transfer to the recipients. Percentages of transgenic blastocyst production were 22.3 and 33.1% for adult and fetal cell lines, respectively. After transfer of cloned and transgenic blastocysts, 28.6 and 36.4% of the recipients were confirmed pregnant and 75 and 33.3% of the pregnancies resulted in the delivery of viable offspring, respectively. To our knowledge, this is the first report of successful live and survived birth of cloned and transgenic offspring through a whole procedure of in vitro oocyte maturation and embryo development to the blastocyst stage, and in this study the in vitro efficiencies of cloned and transgenic embryo production were higher than the available reports.
Cloning (programming)
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