A Highly Efficient Method for Porcine Cloning by Nuclear Transfer Using <i>In Vitro</i>-Matured Oocytes

Walker, S.; Shin, Tae Young; Zaunbrecher, Gretchen M.; Romano, Juan E.; Johnson, Greg A.; Bazer, Fuller W.; Piedrahita, Jorge A.

doi:10.1089/153623002320253283

Cited by 140 publications

(104 citation statements)

References 13 publications

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“…We and authors of another study reported that phenotypic instability in swine resulted from scNT Archer et al, 2003;Park et al, 2004Park et al, , 2005. However, other groups have observed few problems (Betthauser et al, 2000;De Sousa et al, 2002;Walker et al, 2002;Yin et al, 2002). In cattle, the donor cell type was found to be responsible for some of the abnormal phenotypes that were observed, as cells derived from adults resulted in more abnormalities (Heyman et al, 2002).…”

Section: Discussionmentioning

confidence: 80%

Serial cloning of pigs by somatic cell nuclear transfer: Restoration of phenotypic normality during serial cloning

Cho

Kim

Park

et al. 2007

Developmental Dynamics

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Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce firstgeneration (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age-and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development. Developmental Dynamics 236:3369 -3382, 2007.

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Section: Discussionmentioning

confidence: 80%

Serial cloning of pigs by somatic cell nuclear transfer: Restoration of phenotypic normality during serial cloning

Cho

Kim

Park

et al. 2007

Developmental Dynamics

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“…Since the first cloned pigs were generated using somatic cell cloning [4], substantial improvement for this technique has been realized [5,6]. However, the efficiency of pig cloning has been lower than that of other domestic animals, with only 1%-5% of the embryos reconstructed by nuclear transfer surviving to term [7,8]. Apoptosis, a type of programmed cell death, is a physiological process occurring spontaneously during normal preimplantation embryo development [9].…”

Section: Introductionmentioning

confidence: 99%

Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro

Ren

Lü

et al. 2010

J Assist Reprod Genet

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Purpose The purpose of this study was to investigate the relationship of porcine somatic cell nuclear transfer (SCNT) embryo developmental competence with embryonic cell apoptosis and DNA methylation. Methods The apoptotic incidence was examined via comet assay, and the mRNA expression of genes implicated in apoptosis (Bcl-2) and DNA methylation (Dnmt1, Dnmt3a) was determined using real-time RT-PCR. Results Comet assay showed that the SCNT embryos exhibited significantly higher apoptotic rate at 2-cell stage (8.3% versus 2.1%, P<0.05), 16-cell stage (27.3% versus 19.2%, P<0.05) and morula (37.5% versus 26.9, P<0.05) compared with IVF embryos. Compared with IVF embryos, a higher Bcl-2 mRNA expression pattern was observed in SCNT embryos before the 8-cell stage and differed significantly at 2-and 4-cell stages (P<0.05). After the 16-stage, Bcl-2 mRNA expression pattern became significantly lower in SCNT group (P <0.05). The relative expression level of Dnmt1 mRNA showed a higher expression level in oocytes, then sharply decreased and started to increase slightly after the 8-cell (IVF embryos) or 16-cell stage (SCNT embryos). Dnmt1 mRNA expression in IVF embryos appeared to have been lower than that of SCNT group before 16-cell stage embryos, especially at 4-and 8-cell stages (P<0.05). Although a trend for a similar increase of Dnmt3a expression was observed in IVF and SCNT embryos after 8-cell embryos, SCNT group resulted in much higher Dnmt3a mRNA abundance compared with the IVF group, particularly after 16-cell embryos (P<0.05).Conclusions The results showed that low efficiency of porcine SCNT technology may be associated with either embryonic apoptosis or incomplete reprogramming of donor nuclear caused by abnormal Dnmts mRNA expression.

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“…Nuclear transfer was performed by Viagen, Inc. (Austin, TX) as previously described (65). Briefly, ATM-targeted PFF were collected in salt-buffered NCSU-23 containing 10% fetal calf serum and transferred into the oocytes.…”

Section: Nuclear Transfermentioning

confidence: 99%

A novel porcine model of ataxia telangiectasia reproduces neurological features and motor deficits of human disease

et al. 2015

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Ataxia telangiectasia (AT) is a progressive multisystem disorder caused by mutations in the AT-mutated (ATM) gene. AT is a neurodegenerative disease primarily characterized by cerebellar degeneration in children leading to motor impairment. The disease progresses with other clinical manifestations including oculocutaneous telangiectasia, immune disorders, increased susceptibly to cancer and respiratory infections. Although genetic investigations and physiological models have established the linkage of ATM with AT onset, the mechanisms linking ATM to neurodegeneration remain undetermined, hindering therapeutic development. Several murine models of AT have been successfully generated showing some of the clinical manifestations of the disease, however they do not fully recapitulate the hallmark neurological phenotype, thus highlighting the need for a more suitable animal model. We engineered a novel porcine model of AT to better phenocopy the disease and bridge the gap between human and current animal models. The initial characterization of AT pigs revealed early cerebellar lesions including loss of Purkinje cells (PCs) and altered cytoarchitecture suggesting a developmental etiology for AT and could advocate for early therapies for AT patients. In addition, similar to patients, AT pigs show growth retardation and develop motor deficit phenotypes. By using the porcine system to model human AT, we established the first animal model showing PC loss and motor features of the human disease. The novel AT pig provides new opportunities to unmask functions and roles of ATM in AT disease and in physiological conditions. † These authors contributed equally.

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A Highly Efficient Method for Porcine Cloning by Nuclear Transfer Using In Vitro-Matured Oocytes

Cited by 140 publications

References 13 publications

Serial cloning of pigs by somatic cell nuclear transfer: Restoration of phenotypic normality during serial cloning

Serial cloning of pigs by somatic cell nuclear transfer: Restoration of phenotypic normality during serial cloning

Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro

A novel porcine model of ataxia telangiectasia reproduces neurological features and motor deficits of human disease

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