scholarly journals Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts

Nature ◽  
2005 ◽  
Vol 439 (7073) ◽  
pp. 212-215 ◽  
Author(s):  
Alexander Meissner ◽  
Rudolf Jaenisch
Keyword(s):  
Nuclear Transfer ◽  
Es Cells

Animal Embryonic Stem (ES) Cells: Self-renewal, Pluripotency, Transgenesis and Nuclear Transfer

Human Cell ◽  
2008 ◽  
Vol 17 (3) ◽  
pp. 107-116 ◽  
Author(s):  
Shigeo SATTO ◽  
Bingbing LIU ◽  
Kazunari YOKOYAMA
Keyword(s):  
Nuclear Transfer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Self Renewal

500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 101
Author(s):  
J. Antony ◽  
F. Oback ◽  
R. Broadhurst ◽  
S. Cole ◽  
C. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone Demethylase ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Donor Cells

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

scholarly journals Differential methylation status of imprinted genes in nuclear transfer derived ES (NT-ES) cells

Genomics ◽  
2009 ◽  
Vol 93 (2) ◽  
pp. 112-119 ◽  
Author(s):  
Gang Chang ◽  
Sheng Liu ◽  
Fengchao Wang ◽  
Yu Zhang ◽  
Zhaohui Kou ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Es Cells ◽  
Methylation Status ◽  
Differential Methylation ◽  
Imprinted Genes

scholarly journals Human therapeutic cloning, pitfalls and lack luster because of rapid developments in induced pluripotent stem cell technology

2014 ◽  
Vol 8 (1) ◽  
pp. 5-10
Author(s):  
Song Hua ◽  
Henry Chung ◽  
Kuldip Sidhu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Pluripotent Stem Cells ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Therapeutic Cloning ◽  
Induced Pluripotent

AbstractBackground: Therapeutic cloning is the combination of somatic cell nuclear transfer (SCNT) and embryonic stem cell (ES) techniques to create specific ES cells that match those of a patient. Because ES cells derived by nuclear transfer (SCNT ES cells) are genetically identical to the donor, it will not generate rejection by the host’s immune system and thus therapeutically may be more acceptable. Induced pluripotent stem cells (iPS) are a type of pluripotent stem cell artificially derived from an adult somatic cell by inducing a forced expression of a set of specific pluripotent genes. In the past few years, rapid progress in reprogramming and iPS technology has been made, and it seems to shadow any progress made in SCNT programs.Objective: This review compares the application perspective of SCNT with that of iPS in regenerative medicine.Methods:We conducted a literature search using the MEDLINE (PubMed), Wiley InterScience, Springer, EBSCO, and Annual Reviews databases using the keywords “iPS”, “ES”, “SCNT” “induced pluripotent stem cells”, “embryonic stem cells”, “therapeutic cloning”, “regenerative medicine”, and “somatic cell nuclear transfer”. Only articles published in English were included in this review.Results: These two methods both have advantages and disadvantages. Nevertheless, by using SCNT to generate patient-specific cell lines, it eliminates complications by avoiding the use of viral vectors during iPS generation. Success in in vitro matured eggs from aged women and even differentiation of oocytes from germ stem cells will further enhance the application of SCNT in regenerative medicine.Conclusion: Human SCNT may be an appropriate mean of generating patient stem cell lines for clinical therapy in the near future.

scholarly journals Direct Production of Gene-targeted Mice from ES Cells by Nuclear Transfer and Gene Transmission to their Progeny.

2002 ◽  
Vol 51 (4) ◽  
pp. 375-381 ◽  
Author(s):  
Nobuhiro SHIMOZAWA ◽  
Yukiko ONO ◽  
Kaori MUGURUMA ◽  
Kyoji HIOKI ◽  
Yoshihiko ARAKI ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Es Cells ◽  
Direct Production

Generation of embryonic stem cells: limitations of and alternatives to inner cell mass harvest

2008 ◽  
Vol 24 (3-4) ◽  
pp. E4 ◽  
Author(s):  
Sunit Das ◽  
Michael Bonaguidi ◽  
Kenji Muro ◽  
John A. Kessler
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Ethical Considerations ◽  
Pluripotent Cells ◽  
Mammalian Embryo ◽  
Induced Pluripotency ◽  
Early Mammalian Embryo

✓ Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the early mammalian embryo. Because of their plasticity and potentially unlimited capacity for self-renewal, ES cells have generated tremendous interest both as models for developmental biology and as possible tools for regenerative medicine. This excitement has been attenuated, however, by scientific, political, and ethical considerations. In this article the authors describe somatic cell nuclear transfer and transcription-induced pluripotency, 2 techniques that have been used in attempts to circumvent the need to derive ES cells by the harvest of embryonic tissue.

scholarly journals 57 INVESTIGATION OF CYNOMOLGUS MONKEY (MACACA FASCICULARIS) FETUS FIBROBLAST CELL NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 178
Author(s):  
J. Narita ◽  
H. Tsuchiya ◽  
T. Takada ◽  
R. Torii
Keyword(s):  
Embryo Development ◽  
Cynomolgus Monkey ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Fibroblast Cells ◽  
Post Injection ◽  
Embryo Production ◽  
Cell Stage ◽  
Donor Cells ◽  
Whole Mount

Use of nuclear transfer (NT) in the cynomolgus monkey to establish tailor-made ES cells with the final goal of cloned embryo production was investigated. Activation stimulus conditions previously confirmed in parthenote production were used. Injection method NT was conducted using cynomolgus monkey fetus fibroblast cells in order to investigated the time it takes, from injection to activation, to reprogram the donor nuclei. Oocytes were collected under laparoscopic observation from mature cynomolgus monkeys 40 h after hCG (400 IU/kg) administration 9 days after follicle stimulation by i.m. injection of FSH (25 IU/kg). Donor cells, 40-day-old fetus fibroblast cells, were cultivated and synchronized at G0/G1 phase. After mature (MII) oocytes were enucleated using a Piezo-drive unit, donor cells were injected. At 2 h (Experiment 1, E1) and 4 h (Experiment 2, E2) after donor cell injection, activation was carried out by 2-min treatment with ionomycin and cultivation by 6-dimethylaminopurine for 4 h. As a control, parthenote production was carried out under the same activation conditions as NT. After activation, in vitro culture was carried out for about 9 days under conditions of 38°C, 5% CO2, and 5% O2. Whole-mount specimens of NT embryos were made immediately post-injection, 2 and 4 h post-injection, and 2 h after activation. Pronuclear formation (PN) and cleavage rates of NT embryos were 82.1% and 95.7% for E1, and 53.8% and 92.8% for E2, respectively. Control PN and cleavage rates were each 100%. Subsequent embryo development arrested at the 6-cell stage (8.7%) in E1 and 5-cell stage (7.1%) in E2 but proceeded to blastocyst stage (27.3%) in the control. For whole mount specimens, donor nuclei caused premature chromosome condensation in enucleated oocyte cytoplasm, and decondensation due to activation was seen, so injected donor nuclei reconstruction had occurred. No difference was seen between E2 and E1 embryo development and whole mount specimens, but E1 PN rate was clearly higher than that of E2. So 2 h of reprogramming time is more appropriate than 4 h. In this study, most NT embryos arrested at the 4-cell stage. These results suggest that development did not proceed beyond MET (maternal-embryonic transition) which is believed to occur between the 4- and 8-cell stage in cynomolgus monkey. Further study will be necessary to find the condition that completely reprograms injected donor nuclei for cloned embryo production. Table 1. Development of cynomolgus monkey fibroblast nuclear transfer embryos This work was supported by grants from the ministry of Education, Science, Sports, and Culture (13358014, 14380382).

98 HETEROCHROMATIN REPROGRAMMING IN MOUSE EARLY DEVELOPMENT

2009 ◽  
Vol 21 (1) ◽  
pp. 149
Author(s):  
T. Wongtawan ◽  
J. Taylor ◽  
I. Wilmut ◽  
S. Pennings
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Nuclear Transfer ◽  
Technical Assistance ◽  
Es Cells ◽  
Cell Nuclei ◽  
Mouse Development ◽  
Fluorescent Intensity ◽  
Chromatin Architecture ◽  
Somatic Nuclear Transfer

Heterochromatin is essential for epigenetic gene silencing and nuclear chromatin architecture. Early mouse development is accompanied by dynamic epigenetic changes and heterochromatin restructuring in the cell nuclei of cleavage stage embryos. We have previously shown that disruption of heterochromatin markers such as DNA methylation following somatic nuclear transfer causes developmental failure (Beaujean N et al. 2004 Biol. Reprod. 71, 185–193). The aim of the present study was to investigate the transitions and maturation of heterochromatin during normal development to improve nuclear reprogramming technology and understand developmental abnormalities caused by epigenetic alterations in somatic nuclear transfer and assisted reproductive procedures. Mouse pre- and post-implantation embryos and ES cells from B6CBAF1 mice were used in experiments employing the following methodologies. Embryos and ES cells were stained by immunofluorescence to detect heterochromatin proteins and epigenetic markers. Images were captured using laser confocal microscopy. Relative quantification of fluorescent intensity was performed using Zeiss LSM-meta and WCIF-ImageJ software. mRNA of Histone methyltransferase (HMTase), suv39h, suv420h, ehmt, eset, and demethyltransferase (DHMTase), jmjd1a, jmjd2a, jmjd2c were quantified by real-time PCR. siRNA and specific inhibitors were used to study the function of suv39h, ehmt2, jmjd2c genes that may be responsible for heterochromatin reprogramming. Our results demonstrated that H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. The gene expression results showed the dynamic and temporal gene expression of HMTases and DHMTase during development. Inhibition of Ehmt2 and jmjd2c caused preimplantation developmental arrest. Furthermore, we found chromatin modification differences in the heterochromatin of ES cells, ICM and epiblast. We conclude that heterochromatin reprogramming might be essential for development because it may contribute to chromatin architecture, thus influencing gene expression. HMTases and DHTMases could be implicated in the mechanism of heterochromatin reprogramming. It is possible that Ehmt2 and jmjd2c play an important role in preimplantation development by modifying chromatin globally and at the local gene level. This work was supported by a The Royal Thai Studentship. We acknowledge the contributions of technical assistance from T. O’Connor, B. Wongtawan and P. Travers.

24 DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF EMBRYONIC CULTURED CELLS ISOLATED FROM SOMATIC CELL NUCLEAR TRANSFER BLASTOCYSTS

2008 ◽  
Vol 20 (1) ◽  
pp. 92
Author(s):  
X. J. Bai ◽  
J. L. Yu ◽  
M. Murakami ◽  
Y. Zhang ◽  
Y. J. Dong
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cultured Cells ◽  
Es Cells ◽  
Donor Cells ◽  
The Mean ◽  
Development In Vitro ◽  
Chi Square Analysis

Embryonic stem (ES) cells derived from somatic cell nuclear transfer (NT) bovine embryos would increase the utility of the cow as a large animal model for human cell therapy. They would also be useful for studies of cell differentiation. Such cells exhibit full pluripotency, and cloned offspring were obtained from them following a second NT in mice, indicating that the reprogramming that produced pluripotent ES cells could be reversed (Wakayama et al. 2001 Science 292, 740–743). The objective of this study was to examine if there would be any beneficial effects of using somatic cell NT-derived embryonic cultured cells as donors for cloning in cattle. Cloned embryos were produced from a single cell line of bovine fetal fibroblasts (FF) and adult ear-tip cells (AEC) (passages 1 to 5) by NT, as described previously (Dong et al. 2004 Asian–Aust. J. Anim. Sci. 17, 168–173). NT embryos that reached the blastocyst stage were cultured separately to isolate embryonic cultured cells derived from FF (NT-FF) and AEC (NT-AEC) according to previous methods (Dong et al. 2003 Acta Genet. Sin. 30, 114–118). More than 80% of the generated embryonic cultured cells stained positive for alkaline phosphatase. Embryonic cells cultured for 7 to 35 days were used as the donor cells for NT in the NT-FF and NT-AEC groups. Cloned embryos were produced using individual cell lines of FF, AEC, NT-FF, and NT-AEC (passages 1 to 5, putative cell cycle stage of G0 or G1) as donor cells, and their development in vitro is summarized in Table 1. The FF and AEC groups include data from the initial round of NT. The rates of fusion and embryo development were compared by chi-square analysis. Duncan's multiple range test was used to compare the mean cell numbers of blastocysts. The percentage of embryos that developed into blastocysts was significantly higher (P < 0.05) in the FF group than in the AEC group. Interestingly, we observed that the developmental potential in vitro and the mean cell number of blastocysts tended to be higher in the NT-FF and NT-AEC groups than in the FF and AEC groups. A total of 15 and 6 good quality Day 7 embryos in the NT-FF and NT-AEC groups were nonsurgically transferred to 5 and 3 synchronized recipients (2 to 3 embryos/female), respectively. On Day 30 of gestation, 3 (60%) and 1 (33%) females in the NT-FF and NT-AEC groups, respectively, were diagnosed as pregnant via ultrasonography. One (20%) recipient cow in the NT-FF group remained pregnant at Day 60 of gestation, but lost the pregnancy by Day 90. These results suggest that cloning of bovine embryonic cultured cells generated from fetal and adult somatic cells by NT can produce transferable embryos and initiate pregnancies, although none of the pregnancies has developed beyond the first trimester at this time. Table 1. Development in vitro of bovine NT embryos produced from different donor cell types

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