72 LIMITED REPROGRAMMING OF SOMATIC CELL NUCLEI TRANSFERRED INTO MOUSE IMMATURE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 153
Author(s):  
C. Palmieri ◽  
H. Fulka ◽  
J. Fulka, Jr ◽  
P. Loi ◽  
G. Ptak ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cell Nuclei ◽  
Practical Applications ◽  
Functional Changes ◽  
Low Efficiency

Somatic cell nuclear transfer, an important approach for the analysis of certain functional changes in the genome during differentiation and for many practical applications, is in general a low-efficiency procedure, mainly due to a low effectivity in the re-establishment of the developmental program in the reconstructed embryo. The process of reprogramming is, however, poorly understood and some additional studies are clearly necessary. The aim of this study was the ultrastructural and immunofluorescent (B23-nucleophosmin) evaluation of somatic (cumulus) cell nuclei reprogramming after their transfer into intact immature mouse oocytes, kept at the germinal vesicle (GV) stage (dbcAMP) during the whole culture. Control somatic cells and nuclear transfer-reconstructed embryos (1 and 24 h after fusion induced by polyethyleneglycol) were fixed for transmission electron mcroscopy (TEM) in 2.5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated through an ethanol series, and embedded in epoxy resin. Finally, ultrathin sections were stained with uranyl acetate followed by lead citrate. The above reagents were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). In parallel, we have evaluated immunocytochemically the pattern of B23 labelling in intact and reconstructed cells. The samples were fixed in 4% paraformaldehyde, incubated with an antibody against B23 (Santa Cruz, CA, USA) and then with a biotinylated secondary antibody, and detected by fluorescein isothiocyanate (FITC)-coupled Streptavidin (Jackson ImmunoResearch, Cambridgeshire, UK). Mouse cumulus cells (12/12) contain a reticulated fibrillogranular nucleolus. The cell are also positively labeled with the anti-B23 antibody. One hour after fusion, the introduced nuclei displayed shape modifications and nuclear envelope irregularities, whereas the nucleolus still showed the typical fibrillar pattern (19/19). The volume of transferred nuclei remains unchanged. Interestingly, while the oocyte nucleolus remains negative for B23, the nucleoli in transferred somatic cells were always positively labeled (45 cells). After 24 h, the transferred nuclei increased their volume up to two-three times and displayed an irregular shape with nucleoli still possessing the unchanged reticulated pattern (17/17). As in the previous experimental interval, only the somatic cell nucleus remained labeled with the anti-B23 antibody (52 cells). The GV oocyte nucleoli remained unchanged during the whole culture period, exhibiting the typical dense-fibrillar pattern. Our results showed that the immature oocyte cytoplasm possesses a limited remodelling activity. Interestingly, the evident increase in volume of transferred somatic cells indicated some changes but TEM morphology and B23 labeling pattern remained basically unchanged. We cannot, however, exclude the beneficial effect upon reprogramming if these nuclei were subsequently used for the transfer into definitive cytoplasts. This work was supported by ESF STE/05/E004.

47 SUCCESSFULLY PRODUCING CLONED MICE FROM SOMATIC CELLS OF AGED MICE VIA ESTABLISHED ntES CELL LINES

2008 ◽  
Vol 20 (1) ◽  
pp. 104
Author(s):  
E. Mizutani ◽  
T. Ono ◽  
L. Chong ◽  
T. Wakayama
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Chimeric Mice ◽  
Cell Nuclei ◽  
Female Mice ◽  
Aged Mice ◽  
Tail Tip ◽  
Tip Cells

Recent nuclear transfer techniques have enabled us to produce cloned animals from somatic cell nuclei in a variety of animal species and are to date the only way to obtain offspring from infertile animals. Despite very aged mice often showing an infertile phenotype, the decreasing rate of cloning success with increased age makes it almost impossible to produce cloned mice or offspring from these animals. Other studies, however, have demonstrated that ES cell lines have been established from cloned blastocysts through somatic cell nuclear transfer (ntES cells), irrespective of sex, strains, or organs. These cells are subsequently capable of differentiating into all three germ layers in vitro, or even into spermatozoa and oocytes in chimeric mice. Thus, ntES cells have received considerable attention recently in regenerative medicine. Importantly, the success rate of establishing ntES cell lines from cloned blastocysts is ten times higher than that of producing cloned mice, which may allow us to establish ntES cell lines even from such 'unclonable' aged mice. ntES cells also have the potential to be a good donor source for nuclear transfer as they have the same DNA as their donor somatic cells and can indefinitely proliferate in their undifferentiated states. In this study, we attempted to establish ntES cell lines from aged mice and analyze their normality. We then tried to produce cloned mice via nuclear transfer using established ntES cell lines. We obtained donor cells from tail-tip fibroblast cells of BDF1 and BCF1 male and female mice that were over two years old. Following nuclear transfer, we transferred a proportion of the cleaved cloned embryos to pseudopregnant ICR female mice. The remaining embryos were cultured for 72 h, and cloned embryos that developed into morulae or blastocysts were plated on feeder cells. We then examined all established ntES cell lines for normality by Oct4 and Nanog expression using immunofluorescence staining and pluripotency by chimeric mice formation, for which ntES cells were injected into fertilized ICR embryos. Finally, we attempted to produce cloned mice from the nuclei of these ntES cell lines. In each experiment, 25, 37, 73, 63, and 75 cloned embryos from aged mice tail-tip cells were used in attempts to produce cloned mice, and 20, 20, 27, 35, and 40 cloned embryos were used to derive ntES cell lines, respectively. No cloned mice were obtained by direct nuclear transfer of the aged mice tail-tip cells; however, we were successful in establishing ntES cell lines from all experiments, with an establishment rate between 10 and 25%. All established ntES cell lines expressed Oct4 and Nanog and contributed to somatic cells in chimeric mice. Some chimeric mice produced offspring derived from ntES cells after mating. We were also able to produce cloned mice even from a 2-year-old and a 9-month-old BCF1 male mouse by nuclear transfer using ntES cells as donor nuclei. These results clearly show that normal ntES cell lines can be established from infertile, aged mice and this technique can now be used to produce offspring, irrespective of donor conditions.

27 CYTOPLASM OF A GERMINAL VESICLE OOCYTE CAN PROMOTE SOMATIC NUCLEAR REPROGRAMMING IN MICE

2007 ◽  
Vol 19 (1) ◽  
pp. 132
Author(s):  
H.T. Bui ◽  
N. Van Thuan ◽  
S. Kishigami ◽  
S. Wakayama ◽  
T. Hikichi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Chromosome 1 ◽  
Cell Nuclei ◽  
Reconstructed Embryos ◽  
First Polar Body ◽  
Oocyte Cytoplasm ◽  
Series Of Experiments

A number of studies have shown that epigenetic reprogramming is severely deficient in cloned embryos, the majority of which exhibit histone hypermethylation. Xenopus geminal vesicle (GV) oocytes have been reported to have a DNA demethylating activity (Simonsson and Gurdon 2004 Nat. Cell Biol. 6, 984–990). In an attempt to develop a new method for erasing or reprogramming the epigenetic status of the donor cell prior to nuclear transfer, we examined whether the mammalian GV oocyte cytoplasm can demethylate H3-K9 of somatic cell nuclei and improve the quality of reconstructed embryos. In the first series of experiments, cumulus nuclei were injected into enucleated GV oocytes and cultured for various times, 0 h, 3 h, 7 h, 9 h, 11 h, and 16 h, before examining the chromosome morphology of somatic nuclei together with Me-H3-K9. In the second series of experiments, permeabilized cumulus cells were immersed with GV oocyte extract and injected into enucleated MII oocytes. These reconstructed oocytes were activated and cultured until the blastocyst stage. Preparation of oocyte extracts was as follows: 200 GV oocytes were collected and zonae pellucidae removed using Tyrode's solution. These zona-free oocytes were broken down in 5 �L of HEPES medium containing the ATP-generating system (1 mM ATP, 10 mM creatine phosphate, 25 �g mL-1 creatine kinase, 100 �M GTP). Permeabilized cumulus was incubated in oocyte extract for 45 min at 37�C. The demethylation of H3-K9 was analyzed in nuclear transfer embryos at the pronucleus, 2-cell, 8-cell, morula, and blastocyst stages. In 2 experiments, we examined a total of 234 enucleated GV oocytes and 358 enucleated MII oocytes. The results of first experiment showed that histone H3-K9 begins demethylation in the somatic chromosome 1 h after injection into GV oocytes (90%) and, importantly, this state was maintained until the MII-like stage. The donor somatic chromosomes in the enucleated GV oocyte can condense and undergo stages GVI (0–3 h), MI (7 h), and AI-TI (9–11 h), and the MII (16 h)-like stage. These results clearly show that enucleated GV oocytes can carry out spindle assembly and extrude the first polar body. However, some of these chromosomes are located not only on the metaphase plate of the spindle but also on spindle poles or dispersed on the spindle. In the second experiment, reconstructed embryos with GV extract-treated somatic cells showed a strong H3-K9 demethylation that was significantly different to nontreated somatic cells at the blastocyst stage. The demethylation was expressed at a rate comparable to that observed in ICSI embryos. Although there was no increase in the frequency of development at the blastocyst stage, we found an increase in cell number at the blastocyst stage. Our findings show that the GV oocyte cytoplasm has the ability to erase methylation of H3-K9 in somatic nuclei, which suggests that the incorporation of GV mammalian oocyte components may contribute to the reprogramming of somatic cell nuclei.

scholarly journals Developmental potential of cloned mouse embryos reconstructed by a conventional technique of nuclear injection

Reproduction ◽  
2002 ◽  
pp. 197-207 ◽  
Author(s):  
A Rybouchkin ◽  
B Heindryckx ◽  
J Van der Elst ◽  
M Dhont
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Somatic Cells ◽  
Cumulus Cell ◽  
Conventional Technique ◽  
Mouse Embryos ◽  
Full Term ◽  
Cell Nuclei ◽  
Developmental Potential

The fact that most of the advances in mouse cloning by nuclear transfer originate from research in a limited number of laboratories demonstrates the complexity of the reported technologies. The development of alternative and more simple techniques of nuclear transfer may therefore be of interest. Furthermore, the preimplantation biology of cloned mouse embryos originating from somatic cells has not yet been studied in detail. In the present study, a modified conventional injection (mCI) technique for cloning mice from somatic cells is described. The preimplantation development and morphology of the resulting nuclear transfer embryos in comparison with parthenogenetic embryos and embryos obtained by intracytoplasmic sperm injection (ICSI) under comparable conditions was also studied. Finally, the capacity of nuclear transfer embryos for full-term development was investigated. Eighty-nine per cent of oocytes injected with cumulus cell nuclei under mCI conditions survived and formed zygotes. However, the rate of development of these zygotes to the blastocyst stage was significantly lower (29%) than that of ICSI or parthenogenetic zygotes (95 and 92%, respectively). Cloned blastocysts had a significantly lower mean number of cells in the inner cell mass (9) and trophectoderm (52) and a lower inner cell mass:total cell ratio (14%) than did their counterparts (31, 143 and 18% for ICSI and 21, 92 and 18% for parthenogenetic blastocysts, respectively). This correlated with a significantly higher proportion of dead cells in the cloned blastocysts. The poor quality of cloned blastocysts may explain the low rate of full-term fetal development of somatic mouse clones.

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

scholarly journals Lessons Learned from Somatic Cell Nuclear Transfer

2020 ◽  
Vol 21 (7) ◽  
pp. 2314 ◽  
Author(s):  
Chantel Gouveia ◽  
Carin Huyser ◽  
Dieter Egli ◽  
Michael S. Pepper
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Lessons Learned ◽  
Epigenetic Reprogramming ◽  
Area Of Interest ◽  
Legal Implications ◽  
Low Efficiency

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

scholarly journals Differential In Vivo Binding Dynamics of Somatic and Oocyte-specific Linker Histones in Oocytes and During ES Cell Nuclear Transfer

2005 ◽  
Vol 16 (8) ◽  
pp. 3887-3895 ◽  
Author(s):  
Matthias Becker ◽  
Antje Becker ◽  
Faiçal Miyara ◽  
Zhiming Han ◽  
Maki Kihara ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Linker Histone ◽  
Binding Properties ◽  
Paternal Genome ◽  
Linker Histones ◽  
In Vivo Binding

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 139 ◽  
Author(s):  
S. Mitalipov ◽  
Q. Zhou ◽  
J. Byrne ◽  
W.-Z. Ji ◽  
D. Wolf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Lamin A ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Morula Stage

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

2015 ◽  
Vol 27 (1) ◽  
pp. 104
Author(s):  
N. L. Selokar ◽  
M. Saini ◽  
H. Agrawal ◽  
P. Palta ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Frozen Semen ◽  
Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

Kdm6a overexpression improves the development of cloned mouse embryos

Zygote ◽  
2017 ◽  
Vol 26 (1) ◽  
pp. 24-32 ◽  
Author(s):  
Guang-yu Bai ◽  
Si-hang Song ◽  
Yu-wei Zhang ◽  
Xiang Huang ◽  
Xing-wei Huang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Life Science ◽  
Cumulus Cell ◽  
Science Research ◽  
Mouse Embryos ◽  
Rdna Transcription ◽  
X Chromosomes ◽  
Blastocyst Rate

SummarySomatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

scholarly journals Bovine Oocyte Cytoplasm Supports Development of Embryos Produced by Nuclear Transfer of Somatic Cell Nuclei from Various Mammalian Species1

1999 ◽  
Vol 60 (6) ◽  
pp. 1496-1502 ◽  
Author(s):  
Tanja Dominko ◽  
Maissam Mitalipova ◽  
Brad Haley ◽  
Zeki Beyhan ◽  
Erdogan Memili ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Bovine Oocyte ◽  
Cell Nuclei ◽  
Oocyte Cytoplasm
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