38 RABBIT NUCLEAR TRANSFER AND IN VIVO-FERTILIZED EMBRYOS FAIL TO EXPRESS A MOUSE Oct-4 PROMOTER-DRIVEN EGFP REPORTER GENE

2007 ◽  
Vol 19 (1) ◽  
pp. 137 ◽  
Author(s):  
R. Hao ◽  
A. Wuensch ◽  
R. Klose ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Dna Methylation ◽  
Fluorescence Microscopy ◽  
Reporter Gene ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Bovine Embryos ◽  
Egfp Gene ◽  
Gene Construct ◽  
Fetal Fibroblasts

Reprogramming of a donor cell genome during somatic cell nuclear transfer (SCNT) is largely dependent on appropriate expression of 'pluripotency'? genes, such as Oct-4 (POU5F1). Recently, we transfected bovine fetal fibroblasts with GOF18-ΔPE-EGFP, a reporter gene construct for the Oct-4 promoter and assessed the expression of Oct-4 after SCNT (Wuensch et al. 2006 Reprod. Fertil. Dev. 18, 144). Our previous study on DNA methylation reprogramming revealed that rabbit in vivo-fertilized and cloned embryos differ from bovine embryos in respect to this epigenetic modification (Shi et al. 2004 Biol. Reprod. 71, 340–347), suggesting differences in the mechanism of epigenetic reprogramming between these two species. In this study, we tested whether GOF18-ΔPE-EGFP could be used to monitor Oct-4 expression in rabbit cloned embryos. The reporter gene construct included the EGFP gene flanked by a 9-kb fragment of the murine Oct-4 upstream region with a deletion in the proximal enhancer (PE) and a 9-kb fragment containing the nontranscribed murine structural Oct-4 gene. The 21.2-kb fragment GOF18-DPE-EGFP was released from the vector backbone by NotI digestion and purified with QIAquickGel Extraction Kit (Qiagen, Hilden, Germany) after gel electrophoresis. Four stable transfected colonies of rabbit fetal fibroblasts (RFF), none of which exhibited green fluorescence, were used for SCNT. The resulting embryos were examined on Days 2–5 by fluorescence microscopy. To detect endogenous Oct-4 expression, in vivo-fertilized embryos were stained with anti-mouse Oct-3 antibody and then incubated with secondary Alexa 488-conjugated goat anti-mouse antibody. The most prominent endogenous Oct-4 expression was detected in in vivo-fertilized embryos at the morula and blastocyst stages. Depending on the donor cell line used for nuclear transfer, cleavage and blastocyst rates ranged from 56 to 97% and from 33 to 49%, respectively. When a total of 230 cloned embryos at the 2-to 16-cell stages and 93 cloned morulae and blastocycts were examined by fluorescence microscopy, none of the examined embryos exhibited fluorescence signals indicating the lack of Oct-4 promoter activity. Taking into account the fact that both cloned and in vivo-fertilized rabbit embryos have specific patterns of DNA methylation reprogramming, which are different from that of bovine embryos, we injected GOF18-ΔPE-EGFP gene constructs into pronuclei of in vivo-fertilized zygotes. None of the 74 injected embryos, which were examined at the 2-cell to blastocyst stages, showed fluorescence signals. Our results demonstrate that rabbit nuclear transfer and in vivo-fertilized embryos are unable to activate a mouse Oct-4 promoter-reporter construct. Potential reasons include incompatibilities between mouse Oct-4 promoter sequences and rabbit transcription factors as well as specific mechanisms of epigenetic reprogramming in the rabbit. This work was supported by the Bayerische Forschungsstiftung.

259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
B. Mohana Kumar ◽  
H. F. Jin ◽  
J. G. Kim ◽  
S. A. Ock ◽  
H. J. Song ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Dna Hypomethylation ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Donor Cells ◽  
Fetal Fibroblasts

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 �m, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 �m 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P < 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 � 5.2% vs. 65.8 � 5.3% and 69.3 � 4.4%, respectively). Similarly, significantly (P < 0.05) higher blastocyst rates were observed in IVF embryos (27.2 � 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P < 0.05) higher rates of blastocysts (21.3 � 2.2%) than NT embryos from PFF (14.8 � 1.9%). NT embryos from PFF + 5-azaC (33.8 � 4.1) had significantly (P < 0.05) higher total cell numbers than from PFF (24.6 � 3.5), but did not differ in the proportion of apoptotic cells (6.9 � 1.8% and 7.2 � 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 � 3.8, 2.7 � 0.8%, and 53.9 � 3.5, 1.2 � 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

scholarly journals 33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED AT G0/G1 PHASE BY DIFFERENT TREATMENTS

2004 ◽  
Vol 16 (2) ◽  
pp. 139
Author(s):  
S.R. Cho ◽  
W.J. Son ◽  
C.S. Park ◽  
S.Y. Choe ◽  
G.J. Rho
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Group 1

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P<0.05). Blastocyst development of cloned embryos was significantly (P<0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

43 INFLUENCE OF DONOR CELL DIFFERENTIATION ON CLONING EFFICIENCY IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 140
Author(s):  
N. Hornen ◽  
W. A. Kues ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
P. Hassel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Primary Cultures ◽  
Donor Cell ◽  
Stem Cell Population ◽  
High Density Culture ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Normal Offspring

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 � 10.5% (mean � SD) vs. 83.7 � 9.2%; fetal fibroblasts: 64.8 � 17.3% vs. 82.5 � 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 � 3.5% vs. 24.9 � 8.6%). In the second experiment, FSSCs of 3 different sizes (<14 �m, 15–20 �m, >21 �m), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 � 3.1 vs. 91.3 � 10.1 vs. 92.3 � 5.1; cleavage rates: 83.5 � 7.9 vs. 83.1 � 1.6 vs. 83.2 � 5.8; blastocyst rates: 15.3 � 7.9 vs. 17.6 � 6.8 vs. 10.4 � 2.7, respectively). In the third experiment, FSSCs 15–20 �m in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3� 7.3% vs. 86.8 � 5.3%; cleavage rates: 86.1 � 6.7% vs. 80.7 � 5.9%; blastocyst rates: 21.4 � 5.6% vs. 18.4 � 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced.

scholarly journals 36COMPARISON OF THE DEVELOPMENTAL POTENTIAL OF CAPRINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM IN VITRO AND IN VIVO MATURED OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 140
Author(s):  
Y. Echelard ◽  
E. Memili ◽  
S.L. Ayres ◽  
M. O'Coin ◽  
L.H. Chen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Reproductive Tract ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Student’S T

The objective of this study was to compare the development to the blastocyst stage of reconstructed caprine nuclear transfer (NT) embryos derived from two sources of ova. In vivo oocytes were flushed from the oviduct of superovulated donors by exposing the reproductive tract via a small ventral laparotomy. In vitro oocytes were collected from ovaries supplied by an abattoir located in Purdue, IN. Oocytes were aspirated, cultured in maturation medium (M199 +10% goat serum, 3μgmL−1 LH, 3μgmL−1 FSH and 0.22mM sodium pyruvate), and shipped overnight (38°C, air). Donor cell preparation and NT procedures were as previously reported (Behboodi et al., 2001 Theriogenology 55, 254 abst). Donor cells were transfected female fetal fibroblasts that were synchronized by 4 days of serum starvation, followed by a 10-hour exposure to medium containing 10% FCS. Oocytes were enucleated, karyoplast-cytoplast couplets were reconstructed, fused and then activated simultaneously by a single electrical pulse. Couplets containing in vitro oocytes were incubated in the presence of 5μgmL−1 ionomycin after fusion. Fused couplets were co-cultured in TCM199 with 10% FCS and oviductal epithelial cells for 8–10 days (38°C, 5% CO2). Embryos that developed in vitro to the blastocyst stage were surgically transferred to recipients. Pregnancies were confirmed by ultrasonography. One live kid was delivered on Day 150 of gestation via elective C-section. Southern blotting analysis confirmed that it was derived from the transgenic donor cell line. These experiments show that in vivo matured oocytes not only better support caprine NT embryo development to the blastocyst stage, but also can result in live birth (table). Although fusion and cleavage rates were similar in the two groups, development to the blastocyst stage was significantly higher (Student’s t-test) in the group utilizing in vivo-matured oocytes. In conclusion, this is the first live goat produced from goat NT blastocysts developed in vitro. This suggests that in vivo matured oocytes may be superior to oocytes developed in vitro for generating live animals from NT blastocysts. Table 1

scholarly journals 59IN VITRO AND IN VIVO SURVIVAL OF NELORE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH ADULT AND FETAL FIBROBLASTS

2004 ◽  
Vol 16 (2) ◽  
pp. 151
Author(s):  
M.R.B. Mello ◽  
H.V.A. Caetano ◽  
M.S. Padilha ◽  
M.G. Marques ◽  
A.C. Nicacio ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Pregnancy Rates ◽  
Fetal Cells ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Adult Fibroblasts ◽  
Adult Cells

Adult skin and fetal fibroblasts are the most frequently used donor cell types for bovine cloning by nuclear transfer (NT) but there are few reports concerning Nelore cattle. The aim of this study was to evaluate in vitro and in vivo viability of Nelore nuclear transfer embryos reconstructed with Metaphase II oocytes and differentiated somatic cells (adult ear and fetal fibroblasts). Oocytes from ovaries collected at slaughterhouse were matured in vitro for 17h. Enucleation was conducted by aspiration of the first polar body (PB) and small volume of cytoplasm containing metaphase plate. For NT, each nucleus donor cell was inserted under the zona pellucida of each enucleated oocyte and the enucleated oocyte-nuclei donor cell complexes were electrofused (2 pulses of 4KVcm−1 for 20s). After electrical activation, the couplets were incubated in TCM199 plus 7.5% FCS supplemented with cycloheximide (10gmL−1) and cytochalasin D (2.5gmL−1) for 1h and ciclohexemide alone for 4 additional hours. Immediately after activation, reconstructed embryos were co-cultured with granulosa cells in SOF + 5% FCS for 7–9 days. At 7th day of culture, some blastocysts were fixed for counting cells and some transferred into recipients. A total of 377 couplets were reconstructed from fetal and 457 from adult fibroblasts. After electrofusion, 138 (fetal cells) and 166 (adult cells) embryos were incubated, and 24 (17.4%) and 26 (15.7%) reached blastocyst stage, respectively. The blastocyst cell number means were 101.3, 120 and 114.3, respectively, for adult, fetal and IVF (control) embryos. There were no significant differences in the numbers of cells of blastocysts among the groups. After transferring 18 (fetal cells) and 21 (adult cells) blastocysts, pregnancy rates at day 90 were 16.7% (3) and 19% (4). There were no significant differences between pregnancy rates. The first pregnancy from fetal cells delivered a healthy male calf weighing 34kg at Day 290. One of the remaining recipients died with hydrallantois at Day 229 and the other aborted at Day 252. There are four 5-month-pregnancies of adult fibroblast reconstructed embryos. These results indicate that NT embryos produced by fetal and adult fibroblasts of Nelore breed show similar rates of in vitro and in vivo developments. This work was supported by FAPESP (99/07377-3).

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

scholarly journals Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

2018 ◽  
Vol 50 (4) ◽  
pp. 1376-1397 ◽  
Author(s):  
Yanhui Zhai ◽  
Zhiren Zhang ◽  
Hao Yu ◽  
Li Su ◽  
Gang Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Modifications ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Expression Levels ◽  
Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 236
Author(s):  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Marrow Mesenchymal Stem Cells

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

Sign in / Sign up

Export Citation Format

Share Document