Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells

Abstract The completely new strategy of pseudophysiological transcomplementary (transcytoplasmic) activation (PP-TCA) of nuclear-transferred oocytes, which had been derived from pWAPhGH-GFPBsd transfected foetal fibroblast cells, was recently applied to the somatic cell cloning of pigs. It resulted in the considerable enhancing not only the cleavage activity of cultured cloned embryos, but also their morula and blastocyst formation rates as compared to the use of standard simultaneous fusion and electrical activation of reconstituted oocytes (77% vs. 57%, 63% vs. 46% and 40% vs. 27%, respectively). Altogether, the use of cytosolic components descended from heterologous (rabbit) zygotes as the agents for stimulation of porcine clonal cytoplasmic hybrids (cybrids) turned out to be reliable and feasible strategy for the generation of transgenic blastocysts by somatic cell nuclear transfer (SCNT). Furthermore, to our knowledge, no previous study has reported the preimplantation developmental outcome of transgenic nuclear-transferred pig embryos following the PP-TCA that was developed and optimised in our laboratory.

Download Full-text

In vitro development of reconstructed goat oocytes after somatic cell nuclear transfer with fetal fibroblast cells

Small Ruminant Research ◽

10.1016/s0921-4488(03)00014-2 ◽

2003 ◽

Vol 48 (3) ◽

pp. 217-225 ◽

Cited By ~ 12

Author(s):

S.K Das ◽

A.C Majumdar ◽

G Taru Sharma

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Fibroblast Cells ◽

Fetal Fibroblast ◽

In Vitro Development ◽

Vitro Development

Download Full-text

185 NONSURGICAL EMBRYO TRANSFER FOR PRODUCTION OF PIGS BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab185 ◽

2010 ◽

Vol 22 (1) ◽

pp. 251

Author(s):

J.-G. Yoo ◽

M.-R. Park ◽

H.-N. Kim ◽

Y.-G. Ko ◽

J.-Y. Lee ◽

...

Keyword(s):

Somatic Cell ◽

Embryo Transfer ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Blastocyst Stage ◽

Fibroblast Cells ◽

Vitro Fertilization ◽

Miniature Pigs ◽

Donor Cells

Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.

Download Full-text

36 SERIAL SOMATIC CELL NUCLEAR TRANSFER INCREASES PREGNANCY LOSSES IN GOATS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab36 ◽

2017 ◽

Vol 29 (1) ◽

pp. 125

Author(s):

M. Yang ◽

J. Hall ◽

Q. Meng ◽

Z. Fan ◽

I. Polejaeva

Keyword(s):

Dna Methylation ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Pregnancy Loss ◽

Methylation Pattern ◽

Fibroblast Cells ◽

First Polar Body ◽

Significant Difference ◽

Dna Methylation Pattern

Serial cloning by somatic cell nuclear transfer (SCNT) has been successful in several mammalian species. This method can be beneficial for transgenic line expansion or resetting the lifespan of transgenic cells. Previous studies in bovine and porcine have shown a decrease in efficiency over multiple iterations of serial cloning. However, the contradictory data has been reported in mice where no decrease in cloning efficiency was observed after 25 generations of recloning. To our knowledge, no data have been reported investigating the efficiency of serial cloning in goats. The aim of this study was to evaluate whether there is an effect of recloning on goat SCNT efficiency. αMHC-TGF-β1 fetal fibroblast cells (containing transforming growth factor-β under control of a cardiac-specific promoter) were produced by electroporation and used for the first round of SCNT. For serial cloning, we used neonatal fibroblast cells obtained from skin biopsies used as nuclear donors. These cells were collected from the transgenic cloned goats generated by the first round of SCNT. Cumulus-oocyte complexes recovered from abattoir-derived ovaries using slicing technique were matured in vitro for 20 to 24 h. The first polar body and metaphase plate were removed from a cumulus cell-free oocyte, and a donor fibroblast cell was subsequently transferred into the enucleated oocyte. Fused embryos were then activated for 5 min in 5 mM ionomycin followed by 4 h in 2 mM DMAP with 5 mg mL−1 cycloheximide. Activated embryos were cultured in G1 medium with 5 mg mL−1 BSA for 12 h, followed by surgical transfer into the oviducts of recipients synchronized to show oestrus within 12 h of SCNT. In total, 592 and 395 embryos were transferred to 37 and 25 recipient goats, respectively, for the first and second round of SCNT. Pregnancy rate, rate of pregnancy loss, and term rate were analysed by Chi-squared with a 2-tailed P-value. No significant difference was observed in Day 40 pregnancy rates (32.4 v. 36%) and term rates (32.4 v. 20%) between the first round of cloning and the successive recloning. However, the rate of pregnancy losses was significantly greater in recloning group (P < 0.05), with 4 out of 9 pregnancies lost between Day 40 of gestation and term, whereas no pregnancy losses were observed after Day 40 of gestation in the first-round cloning group. The greater pregnancy loss in the recloning procedure might be caused by accumulation of epigenetic errors resulting from incomplete reprogramming. We are assessing the DNA methylation pattern of differentially methylated regions (DMR) of 2 paternally imprinted genes (H19 and IGF2R) in the cloned and recloned goats and expect to see a difference in their imprinted gene DNA methylation pattern, which could explain the greater rate of pregnancy loss in recloned goats.

Download Full-text

Analysis of Heterogeneous Mitochondria Distribution in Somatic Cell Nuclear Transfer Porcine Embryos

Microscopy and Microanalysis ◽

10.1017/s1431927608080896 ◽

2008 ◽

Vol 14 (5) ◽

pp. 418-432 ◽

Cited By ~ 7

Author(s):

Zhisheng Zhong ◽

Yanhong Hao ◽

Rongfeng Li ◽

Lee Spate ◽

David Wax ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Mouse Fibroblast ◽

Mitochondrial Morphology ◽

Fibroblast Cells ◽

Developmental Potential ◽

Donor Cells

AbstractWe previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro–fertilized (IVF) embryos (Katayama, M., Zhong, Z.-S., Lai, L., Sutovsky, P., Prather, R.S. & Schatten, H. (2006). Dev Biol299, 206–220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Download Full-text

299 EPIGENETIC REPROGRAMMING OF PORCINE FIBROBLAST CELLS INDUCED BY STURGEON'S OOCYTE EXTRACT

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab299 ◽

2011 ◽

Vol 23 (1) ◽

pp. 247

Author(s):

S. Y. Kim ◽

S. H. Park ◽

M. R. Lee ◽

H. J. Eun ◽

T. S. Kim ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Somatic Cells ◽

Direct Conversion ◽

Epigenetic Reprogramming ◽

Fibroblast Cells ◽

Differentiated Cells ◽

Undifferentiated State ◽

Medical Benefits

The direct conversion of differentiated cells into undifferentiated or pluripotent cells would present scientific and medical benefits because of the potential for customized transplantation therapy. Although somatic cell nuclear transfer is one powerful way to fully reprogram somatic cells into a pluripotent state with the aid of oocyte or egg cytoplasm, the therapeutic applications of this approach have been hindered by technical complications as well as ethical objections. An alternative strategy for epigenetic reprogramming of differentiated cells into pluripotent status is desperately required. We have developed a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte extract to porcine fibroblast cells ex ovo. Porcine fibroblasts were permeabilized by 4 μg mL–1 of digitonin for 2 min at 4°C and then incubated in sturgeon’s oocyte extract for 5 h at 15 to 18°C followed by resealing of the cell membrane. We found that the sturgeon’s oocyte extract induced the reduction of overall levels of tri-methylation at lysine 9 of histone H3 (H3K9Me3), which might be related to preservation of DNA methylation in fully differentiated cells, of porcine fibroblast cells. However, permeabilized porcine fibroblasts after treatment with the extract were increasingly acetylated at lysine 9 on histone 3 (H3K9Ac), which might be associated with expression of pluripotency genes. In addition, the cells treated with the extract showed up-regulation of Oct3/4, Sox2, and Nanog gene expression. When somatic cell nuclear transfer embryos reconstructed by using the treated donor cells were transferred into surrogates, the pregnancy rate was slightly high. These results showed that sturgeon’s oocyte extract can reprogram porcine somatic cells into undifferentiated status. Further work needs to exploit the epigenetic reprogramming of differentiated cells into the undifferentiated state using different species oocyte extract.

Download Full-text

34 Recent Advances in Cloning by Somatic Cell Nuclear Transfer in Camelids

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab34 ◽

2018 ◽

Vol 30 (1) ◽

pp. 156

Author(s):

N. A. Wani ◽

V. S. Binoy ◽

S.-B. Hong

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Skin Fibroblast ◽

Bactrian Camel ◽

Dromedary Camel ◽

Fibroblast Cells ◽

Developmental Potential ◽

Bactrian Camels

In addition to its application for production of elite males, racing champions, animals with the highest potential for milk production, and the prized beauty camels, somatic cell nuclear transfer (SCNT) can be utilised for the conservation of endangered wild Bactrian camels and vicunas by using the technique of interspecies SCNT (iSCNT). In the present study, embryos were reconstructed by using skin fibroblast cells from a Bactrian camel (Camelus bactrianus), a llama (Llama glama), and a dromedary camel (Camelus dromedarius) as donor karyoplasts and dromedary oocytes as recipient cytoplasts to evaluate in vitro and in vivo developmental potential of these embryos. Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated oocytes. The fibroblast cell and recipient cytoplasm were fused by 2 DC pulses of 100V for 15 µs each. Reconstructs were activated 1 to 1.5 h post-fusion with 5 µM ionomycin, followed by exposure to 6-DMAP for 4 h. The activated oocytes were then cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. A lower blastocyst production rate was observed when llama fibroblast cells were used as karyoplast (13.3 ± 3.3) compared with those obtained from the embryos reconstructed with Bactrian (34.4 ± 3.9) and dromedary (32.2 ± 6.2) fibroblast cells; however, no difference was observed in their cell numbers. Out of 26 and 20 blastocysts from reconstructs with Bactrian and dromedary fibroblast cells, transferred to 23 and 12 synchronized dromedary recipients, 3 and 2 pregnancies were achieved at Day 60, respectively. One pregnancy from each group reached term and both (a Bactrian and a dromedary) calves were delivered normally after completing the gestation period. We demonstrated birth of a Bactrian calf conceived from the reconstructed embryo by iSCNT using the somatic cell from a Bactrian camel and enucleated dromedary oocyte. The present study also demonstrated that dromedary camel can be used as a surrogate to carry these pregnancies to term (Wani et al. 2017 PLoS One 12, e0177800; https://doi.org/10.1371/journal.pone.0177800). This study has opened doors for enhanced multiplication and preservation of the wild Bactrian camels, which are threatened with extinction, being the eighth most endangered large mammal on the planet.

Download Full-text