scholarly journals Production of Genetically Modified Porcine Blastocysts by Somatic Cell Nuclear Transfer: Preliminary Results Toward Production of Xenograft-Competent Miniature Pigs

2010 ◽  
Vol 56 (6) ◽  
pp. 630-638 ◽  
Author(s):  
Takehiro HIMAKI ◽  
Satoshi WATANABE ◽  
Haiei CHI ◽  
Mitsutoshi YOSHIDA ◽  
Kazuchika MIYOSHI ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Genetically Modified ◽  
Preliminary Results ◽  
Miniature Pigs

scholarly journals Production of human apolipoprotein(a) transgenic NIBS miniature pigs by somatic cell nuclear transfer

2016 ◽  
Vol 65 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Yoshiki Shimatsu ◽  
Wataru Horii ◽  
Tetsuo Nunoya ◽  
Akira Iwata ◽  
Jianglin Fan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Miniature Pigs ◽  
Human Apolipoprotein ◽  
Apolipoprotein A

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 153-159 ◽  
Author(s):  
Satoshi Sugimura ◽  
Manabu Kawahara ◽  
Takuya Wakai ◽  
Ken-ichi Yamanaka ◽  
Hiroshi Sasada ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Uniform Distribution ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Polar Body ◽  
Formation Rate ◽  
Developmental Competence ◽  
Miniature Pigs ◽  
Artificial Activation

SummaryIn many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 μg/ml) and CD (2.5 μg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 μg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 μg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 μg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 μg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 μg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.

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

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.

Production of Transgenic Cloned Miniature-Pigs with Membrane-Bound Human Fas Ligand (FasL) by Somatic Cell Nuclear Transfer.

2010 ◽  
Vol 83 (Suppl_1) ◽  
pp. 701-701 ◽  
Author(s):  
Ki Myung Choi ◽  
Dong Il Jin ◽  
Seung Pyo Hong ◽  
Ji Yeon Yoo ◽  
Soo Hyun Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fas Ligand ◽  
Miniature Pigs ◽  
Human Fas Ligand ◽  
Membrane Bound

24 TARGETED DISRUPTION OF ATAXIA-TELANGIECTASIA MUTATED GENE IN MINIATURE PIGS BY SOMATIC CELL NUCLEAR TRANSFER

2012 ◽  
Vol 24 (1) ◽  
pp. 124
Author(s):  
Y. J. Kim ◽  
K. S. Ahn ◽  
M. J. Kim ◽  
J. S. Ahn ◽  
J. H. Ryu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Ataxia Telangiectasia ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
The United States ◽  
Ataxia Telangiectasia Mutated ◽  
Targeted Disruption ◽  
Miniature Pigs ◽  
Progressive Cerebellar Ataxia ◽  
Fetal Fibroblasts

Ataxia-telangiectasia (A-T) is a genetic disease caused by germline biallelic mutation in the ataxia-telangiectasia mutated gene (ATM) that results in partial or complete loss of ATM expression or activity. A diverse phenotype of the disease includes progressive cerebellar ataxia, oculocutaneous telangiectasias, radiation hypersensitivity, increased cancer incidence, immunodeficiency and chromosomal instability. The frequency of A-T in the United States and United Kingdom has been estimated to be 1:40 000. Heterozygous carriers may also have predisposition to diverse cancers. Although ATM-deficient mice have been produced, none reflects the extent of neurological abnormalities involving the loss of Purkinje cells of the cerebellum seen in patients. Hence, miniature pigs, which are anatomically and physiologically closer to humans, might serve as an alternative model for human A-T. In the present study, we attempted a targeted disruption of ATM in miniature pigs by somatic cell nuclear transfer. Most A-T patients possess mutated ATM with truncation, resulting in prematurely terminated ATM proteins that are highly unstable. To induce the truncation of ATM, we disrupted exon 59 region of ATM that has been known to be critical for ATM kinase activity. Miniature pig fetal fibroblasts were transfected with ATM-targeting vector and treated by neomycin for 2 weeks. A total of 139 colonies were screened by PCR and 3 among them were identified as homologous recombinants with monoallelic disruption of ATM (targeting efficiency = 2.1%). One of the 3 colonies was chosen and used for subsequent nuclear transfer. In total, 611 nuclear transfer embryos reconstructed with ATM-targeted fetal fibroblasts were transferred into 5 surrogate gilts. Two gilts became pregnant and developed to term with a total of 5 live piglets delivered. Being analysed by PCR, all piglets born were found to be ATM gene-targeted. Because conventional murine models of human A-T have provided only limited insights into therapies and pharmacological treatments, the generation of miniature pigs with disrupted ATM will allow new opportunities to more precisely understand A-T and to accelerate discovery of strategies for prevention and treatment of the disease. In addition, such animals may be utilised in studying cancer with respect to the involvement of ATM in cell cycle arrest, DNA repair and apoptosis.

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