Cloning of Homozygous a1,3-Galactosyltransferase Gene Knock-Out Pigs by Somatic Cell Nuclear Transfer

Matsunari, Hitomi; Watanabe, Masaru; Umeyama, Kazuhiro; Nakano, K.; Ikezawa, Yuka; Kessler, Mayuko Kurome Barbara; Wolf, Eckhard; Miyagawa, Shuji; Nakauchi, Hiromitsu; Nagashima, Hiroshi

doi:10.5772/29267

Cited by 5 publications

(10 citation statements)

References 22 publications

(42 reference statements)

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“…The developmental potential of cloned embryos has been reported to improve dramatically by treatment with HDACi (Zhao et al. 2009), and we have confirmed this effect (Matsunari et al. 2012).…”

Section: Disease Model Pigssupporting

confidence: 80%

“…Thus, for example, Umeyama et al (2009) reported marked differences in non-fasting blood glucose levels and longevities among clones of their diabetes model pig, which might reflect variations in the epigenetic status among the cloned siblings. The developmental potential of cloned embryos has been reported to improve dramatically by treatment with HDACi (Zhao et al 2009), and we have confirmed this effect (Matsunari et al 2012). However, we also found that the phenotypes of cloned pigs developed from SCNT embryos treated with HDACi showed as wide variation as nontreated clones (H Matsunari and H Nagashima, unpublished data).…”

Section: Disease Model Pigssupporting

confidence: 79%

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Advancing Pig Cloning Technologies Towards Application in Regenerative Medicine

Nagashima

Matsunari

Nakano

et al. 2012

Reprod Domestic Animals

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Contents Regenerative medicine is expected to make a significant contribution by development of novel therapeutic treatments for intractable diseases and for improving the quality of life of patients. Many advances in regenerative medicine, including basic and translational research, have been developed and tested in experimental animals; pigs have played an important role in various aspects of this work. The value of pigs as a model species is being enhanced by the generation of specially designed animals through cloning and genetic modifications, enabling more sophisticated research to be performed and thus accelerating the clinical application of regenerative medicine. This article reviews the significant aspects of the creation and application of cloned and genetically modified pigs in regenerative medicine research and considers the possible future directions of the technology. We also discuss the importance of reproductive biology as an interface between basic science and clinical medicine.

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Section: Disease Model Pigssupporting

confidence: 80%

Section: Disease Model Pigssupporting

confidence: 79%

Advancing Pig Cloning Technologies Towards Application in Regenerative Medicine

Nagashima

Matsunari

Nakano

et al. 2012

Reprod Domestic Animals

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“…Skin fibroblast cells were isolated from female GalT KO pigs as previously described [ 25 ]. Male fetal fibroblast cells carrying homozygous GalT KO [ 25 , 30 ] were seeded onto type I collagen-coated dishes or plates (Asahi Glass, Tokyo, Japan) and cultured in MEMα (Life Technologies) supplemented with 15% fetal bovine serum (FBS) (Bovogen Biologicals Pty, Victoria, Australia) and 1% antibiotic-antimycotic solution (Life Technologies) in a humidified atmosphere containing 5% CO 2 at 37 C. The fibroblasts were cultured to 70–90% confluency, washed twice with D-PBS(-) (Life Technologies) and treated with 0.05% trypsin-EDTA (Life Technologies) to collect the isolated cells. The cells (4 × 10 5 ) were then resuspended in a mix containing 40 μl of R buffer (supplied as part of the Neon Transfection System, Life Technologies) and 2 μl of ZFN- or TALEN-encoding mRNA solution (400 ng/μl).…”

Section: Methodsmentioning

confidence: 99%

“…We chose the cytidine monophospho- N -acetylneuraminic acid hydroxylase ( CMAH ) gene as a target gene. We previously reported the generation of GalT KO pigs, in which the α-galactosyl (α-Gal) epitope (Galα1-3Galβ1-4GlcNAc-R) mediating xenograft rejection was removed [ 9 , 25 ]. It has also been suggested that removal of the other xeno-epitope, the Hanganutziu-Deicher (H-D) antigen, is also necessary [ 26 , 27 ].…”

mentioning

confidence: 99%

Generation of α1,3-galactosyltransferase and cytidine monophospho-<i>N</i>-acetylneuraminic acid hydroxylase gene double-knockout pigs

Miyagawa

Matsunari

Watanabe

et al. 2015

Journal of Reproduction and Development

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Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

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“…Through the use of genetic modification of pigs, some immunologic challenges have been addressed and considerable progress has been made to alleviate biological incompatibilities between pig and humans [1–4]. However, against the backdrop of these promising developments, there is the inherent risk that porcine endogenous retroviruses (PERV) may be transmitted from the transplanted pig tissue to the human recipient, and this risk has impeded development of xenotransplantation products.…”

Section: Introductionmentioning

confidence: 99%

Susceptibility of porcine endogenous retrovirus to anti‐retroviral inhibitors

Argaw

Colon-Moran

Wilson

2016

Xenotransplantation

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Background Porcine endogenous retrovirus (PERV) is an endogenous retrovirus that poses a risk of iatrogenic transmission in the context of pig-to-human xenotransplantation. The lack of a means to control PERV infection in the context of pig-to-human xenotransplantation is a major concern in the field. In this study, we set out to evaluate the ability of currently licensed anti-HIV drugs, and other types of anti-retroviral compounds, to inhibit PERV infection in vitro. Methods We used target cells stably expressing one of the known PERV viral receptors, an infectious molecular clone, PERV-A 14/220, and at least one drug from each class of anti-retroviral inhibitors as well as off-label drugs shown to have anti-viral activities. The susceptibility of PERV-A 14/220 LacZ to the anti-retroviral drugs was determined from infected cells by histochemical staining. Results We extend the results of previous studies by showing that, in addition to raltegravir, dolutegravir is found to have a potent inhibitory activity against PERV replication (IC50 8.634 ±0.336 and IC50 3.06 ± 0.844 nm, respectively). The anti-HIV drug zidovudine (AZT) showed considerable anti-PERV activity with IC50 of 1.923 ±0.691 μm as well. Conclusions The study results indicate that some of the licensed antiretroviral drugs may be useful for controlling PERV infection. However, the efficacy at nanomolar concentrations put forward integrase inhibitors as a drug that has the potential to be useful in the event that xenotransplantation recipients have evidence of PERV transmission and replication.

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Cloning of Homozygous a1,3-Galactosyltransferase Gene Knock-Out Pigs by Somatic Cell Nuclear Transfer

Cited by 5 publications

References 22 publications

Advancing Pig Cloning Technologies Towards Application in Regenerative Medicine

Advancing Pig Cloning Technologies Towards Application in Regenerative Medicine

Generation of α1,3-galactosyltransferase and cytidine monophospho-<i>N</i>-acetylneuraminic acid hydroxylase gene double-knockout pigs

Susceptibility of porcine endogenous retrovirus to anti‐retroviral inhibitors

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