Serial nuclear transfer improves the developmental potential of mouse embryos cloned from oocytes matured in a protein-free medium

2007 ◽  
Vol 74 (5) ◽  
pp. 560-567 ◽  
Author(s):  
Zhaodai Bai ◽  
Jun Yong ◽  
Tingting Qing ◽  
Jing Cheng ◽  
Wei Shen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Free Medium ◽  
Developmental Potential ◽  
Serial Nuclear Transfer ◽  
Protein Free Medium

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.

Novel method for demonstrating nuclear contribution in mouse nuclear transfer

1998 ◽  
Vol 10 (8) ◽  
pp. 633 ◽  
Author(s):  
Megan Munsie ◽  
Teija Peura ◽  
Anna Michalska ◽  
Alan Trounson ◽  
Peter Mountford
Keyword(s):  
Transgenic Mice ◽  
Nuclear Transfer ◽  
Staining Pattern ◽  
Embryonic Cells ◽  
Simple Method ◽  
Tissue Samples ◽  
Developmental Potential ◽  
Novel Method ◽  
Nuclear Contribution ◽  
Serial Nuclear Transfer

Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring. The unique line of transgenic mice (Zin40) used in this study displays nuclear localised lacZ expression in all cells, including embryonic blastomeres, and demonstrates distinctive blue nuclei when treated with X-gal substrate. This characteristic staining pattern provided an ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos were generated following serial nuclear transfer of metaphase-arrested nuclei from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear transfer blastocysts was confirmed by the generation of live born offspring. Transgenic blastocysts and all tissue samples from fetuses and pups generated by nuclear transfer displayed distinctive blue nuclei when stained with X-gal. This staining pattern was characteristic of the transgenic mice from which the donor nuclei were isolated and clearly confirmed nuclear origin. The use of this marker will also allow the opportunity to investigate the developmental potential of nuclear transfer embryos by examining the contribution of nuclear transfer embryonic cells in chimaeric embryos.

scholarly journals TCL1 promotes blastomere proliferation through nuclear transfer, but not direct phosphorylation, of AKT/PKB in early mouse embryos

2007 ◽  
Vol 15 (2) ◽  
pp. 420-422 ◽  
Author(s):  
M T Fiorenza ◽  
S Torcia ◽  
S Canterini ◽  
A Bevilacqua ◽  
M G Narducci ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Early Mouse

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 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

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