Nuclear transplantation and the control of gene activity in animal development

1970 ◽  
Vol 176 (1044) ◽  
pp. 303-314 ◽  
Keyword(s):  
Nuclear Transfer ◽  
Somatic Cells ◽  
Gene Activity ◽  
Protein Fractions ◽  
Nuclear Transplantation ◽  
Animal Development ◽  
Cytoplasmic Proteins ◽  
Intracellular Movement ◽  
Selective Accumulation ◽  
Structure And Metabolism

The transplantation of nuclei from differentiated or determined somatic cells to enucleated frogs’ eggs consistently leads to a complete and clearly recognizable change of gene activity. Within 1 to 2 h of nuclear transfer, somatic nuclei have come to resemble in structure and metabolism the zygote nuclei of fertilized eggs. The change in gene activity therefore takes place very soon after nuclear transfer and results from an effect of egg cytoplasm. The induced change in gene activity is associated with a selective accumulation of cytoplasmic proteins in transplanted nuclei. Examples are given of various ways in which nuclear transplantation and microinjection can be used to elucidate the intracellular movement of proteins and the effect of known protein fractions on gene activity.

Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolgus monkeys (Macaca fascicularis)

Primates ◽  
2007 ◽  
Vol 48 (3) ◽  
pp. 232-240 ◽  
Author(s):  
Junko Okahara-Narita ◽  
Hideaki Tsuchiya ◽  
Tatsuyuki Takada ◽  
Ryuzo Torii
Keyword(s):  
Nuclear Transfer ◽  
Macaca Fascicularis ◽  
Somatic Cells ◽  
Cynomolgus Monkeys

Cloned rabbits produced by nuclear transfer from adult somatic cells

2002 ◽  
Vol 20 (4) ◽  
pp. 366-369 ◽  
Author(s):  
Patrick Chesné ◽  
Pierre G. Adenot ◽  
Céline Viglietta ◽  
Michel Baratte ◽  
Laurent Boulanger ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Somatic Cells

scholarly journals 57EMBRYO DEVELOPMENT FOLLOWING INTERSPECIES NUCLEAR TRANSFER OF AFRICAN BUFFALO (SYNCERUS CAFFER), BONTEBOK (DAMALISCUS DORCUS DORCUS) AND ELAND (TAUROTRAGUS ORYX) SOMATIC CELLS INTO BOVINE CYTOPLASTS

2004 ◽  
Vol 16 (2) ◽  
pp. 150 ◽  
Author(s):  
M. Matshikiza ◽  
P. Bartels ◽  
G. Vajta ◽  
F. Olivier ◽  
T. Spies ◽  
...  
Keyword(s):  
South Africa ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Critically Endangered ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Cell Stage ◽  
Wildlife Species ◽  
Interspecies Nuclear Transfer

Wildlife conservation requires traditional as well as innovative conservation strategies in order to preserve gene and species diversity. Interspecies nuclear transfer has the potential to conserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where representative cell lines have been established for the wildlife population while numbers were still abundant. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of somatic cells from the African buffalo (Syncerus caffer), bontebok (Damaliscus dorcus dorcus) and eland (Taurotragus oryx) to enucleated domestic cattle (Bos taurus) oocytes. Skin tissue from the three wildlife species were collected by surgically removing approx. 1.0×1.0cm ear skin notches from animals immobilized with a combination of etorphine hydrochloride (M99; South Africa) and azaperone (Stressnil, South Africa). The biopsies were placed into physiological saline and transported to the laboratory at 4°C within 2h, cleaned with chlorohexidine gluconate and sliced finely in Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Baumgarten and Harley 1995 Comp. Biochem. Physiol. 110B, 37–46) and actively growing fibroblast cultures made available for the nuclear transfer process. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578) using slaughterhouse-derived bovine oocytes. Culture was performed in SOFaaci (Vajta et al., 2003 Biol. Reprod. 68, 571–578) medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). Two identical replicates were made with somatic cells of each species. After successful reconstruction, 57, 42 and 48 nuclear transferred and activated buffalo, bontebok and eland embryos were cultured, respectively. All except for 2 buffalo embryos cleaved; 22 (39%) developed to or over the 8-cell stage, and 2 (3.5%) of them to the blastocyst stage. All but 3 bontebok embryos cleaved, 17 (40%) developed to or over the 8-cell stage, but none of them reached the compacted morula or blastocyst stage. Sixteen (33%) of the eland embryos developed to or over the 8-cell stage with one (2%) reaching the blastocyst stage. In conclusion, buffalo, bontebok and eland embryos developed from reconstruction using their respective somatic cells combined with bovine cytoplasts, however, in vitro developmental ability to the blastocyst stage was limited. Additional basic research that establishes the regulative mechanisms involved with early preimplantation development together with optimising nuclear transfer techniques may have the potential to one day play a role in the conservation of critically endangered wildlife species.

scholarly journals Erratum: Production of gene-targeted sheep by nuclear transfer from cultured somatic cells

Nature ◽  
2000 ◽  
Vol 408 (6808) ◽  
pp. 120-120 ◽  
Author(s):  
K. J. McCreath ◽  
J. Howcroft ◽  
K. H. S. Campbell ◽  
A. Colman ◽  
A. E. Schnieke ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Somatic Cells

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.

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

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.

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