α1,3-Galactosyltransferase Gene-Knockout Miniature Swine Produce Natural Cytotoxic Anti-Gal Antibodies

Dor, Frank J. M. F.; Tseng, Yau‐Lin; Cheng, Jane; Moran, Kathleen; Sanderson, Todd M.; Lancos, Courtney J.; Shimizu, Akira; Yamada, Kazuhiko; Awwad, Michel; Sachs, David H.; Hawley, Robert J.; Schuurman, Henk‐Jan; Cooper, D. K. C.

doi:10.1097/01.tp.0000130487.68051.eb

Cited by 81 publications

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References 11 publications

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“…As far as the use of pigs as experimental animals is concerned, miniature pigs are smaller and easier to handle than common domestic pigs, and they have been used in a variety of fields, such as medical and pharmacological research [10,[18][19][20][21][22][23][24][25]. However, compared with common domestic pigs, far less information regarding the production of cloned and genetically modified pigs is available for miniature pigs [10,13,14,[26][27][28][29][30][31].In the present study, we conducted a series of experiments including (i) validation of an estrus synchronization procedure for miniature pig recipients, (ii) examination of the developmental competence of nuclear transfer embryos reconstructed with various nuclear donor cells and (iii) comparison of miniature pigs and common domestic pigs as recipients. The results indicate that it is possible to produce cloned miniature pigs using common domestic pigs as the recipients and to produce genetically modified-cloned miniature pigs with a level of efficiency comparable to that for conventionally cloned miniature pigs.…”

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confidence: 99%

“…54: [156][157][158][159][160][161][162][163] 2008) n recent years, techniques for producing cloned pigs by somatic cell nuclear transfer (SCNT) have been actively utilized to produce genetically modified pigs. A number of cloned pigs have been produced, including those with genes for GFP [1][2][3][4][5][6][7][8], as well as alpha1,3-galactosyltransferase knockout pigs [9][10][11][12][13][14][15][16]. In this manner, genetically modified pigs are being used in a wide variety of biomedical fields, ranging from basic research to organ transplantation.…”

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Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

et al. 2008

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Abstract. Miniature pigs have been recognized as valuable experimental animals in various fields such as medical and pharmaceutical research. However, the amount of information on somatic cell cloning in miniature pigs, as well as genetically modified miniature pigs, is much less than that available for common domestic pigs. The objective of the present study was to establish an efficient technique of cloning miniature pigs by somatic cell nuclear transfer. A high pregnancy rate was achieved following transfer of parthenogenetic (3/3) and cloned (5/6) embryos using female miniature pigs in the early pregnancy period as recipients after estrus synchronization with prostaglandin F2 alpha analog and gonadotrophins. The production efficiency of the cloned miniature pigs using male and female fetal fibroblasts as nucleus donors was 0.9% (2/215 and 3/331, respectively). Cloned miniature pigs were also produced efficiently (7.8%, 5/64) by transferring reconstructed embryos into the uteri of common domestic pigs. When donor cells transfected with the green fluorescent protein (GFP) gene were used in nuclear transfer, the production efficiency of the reconstructed embryos and rate of blastocyst development were comparable to those obtained by non-transfected cells. When transfected cell-derived reconstructed embryos were transferred to three common domestic pig recipients, all became pregnant, and a total of ten transgenic cloned miniature pigs were obtained (piglet production efficiency: 2.7%, 10/365). Hence, we were able to establish a practical system for producing cloned and transgenic-cloned miniature pigs with a syngeneic background. Key words: Embryo transfer, In vitro matured oocyte, Miniature pig, Nuclear transfer, Transgenic (J. Reprod. Dev. 54: [156][157][158][159][160][161][162][163] 2008) n recent years, techniques for producing cloned pigs by somatic cell nuclear transfer (SCNT) have been actively utilized to produce genetically modified pigs. A number of cloned pigs have been produced, including those with genes for GFP [1][2][3][4][5][6][7][8], as well as alpha1,3-galactosyltransferase knockout pigs [9][10][11][12][13][14][15][16]. In this manner, genetically modified pigs are being used in a wide variety of biomedical fields, ranging from basic research to organ transplantation.To date, more than ten breeds of miniature pigs have been established as experimental and companion animals [17]. As far as the use of pigs as experimental animals is concerned, miniature pigs are smaller and easier to handle than common domestic pigs, and they have been used in a variety of fields, such as medical and pharmacological research [10,[18][19][20][21][22][23][24][25]. However, compared with common domestic pigs, far less information regarding the production of cloned and genetically modified pigs is available for miniature pigs [10,13,14,[26][27][28][29][30][31].In the present study, we conducted a series of experiments including (i) validation of an estrus synchronization procedure for miniature pig recipients, (i...

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Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

et al. 2008

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“…Genetically modified pigs such as alpha 1,3-galactosyltransferase-knockout pigs [7][8][9][10][11][12][13][14] and those with a n-3 fatty acid desaturase gene [15] have been produced using serial cloning technology. In this context, multiple generations of clones up to the third generation have been reported in pigs [16,17].…”

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Production Efficiency and Telomere Length of the Cloned Pigs Following Serial Somatic Cell Nuclear Transfer

Kurome

Higuchi

Matsumoto

et al. 2008

Journal of Reproduction and Development

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Abstract. The aim of the present study was to examine the production efficiency of cloned pigs by serial somatic cell nuclear transfer (SCNT) and to ascertain any changes in the telomere lengths of multiple generations of pigs. Using fetal fibroblasts as the starting nuclear donor cells, porcine salivary gland progenitor cells were collected from the resultant first-generation cloned pigs to successively produce second-and third-generation clones, with no significant differences in production efficiency, which ranged from 1.4% (2/140) to 3.3% (13/391) among the 3 generations. The average telomere lengths (terminal restriction fragment values) for the first, second and third generation clones were 16.3, 18.1 and 20.5 kb, respectively, and were comparable to those in age-matched controls. These findings suggest that thirdgeneration cloned pigs can be produced by serial somatic cell cloning without compromising production efficiency and that the telomere lengths of cloned pigs from the first to third generations are normal. Key words: Aging, Pig cloning, Progenitor cell, Somatic cell nuclear transfer (SCNT), Telomere (J. Reprod. Dev. 54: [254][255][256][257][258] 2008) ince the birth of the first cloned sheep, more than 10 animal species have been cloned by SCNT [1]. However, various questions and problems remain with regard to production of somatic cell clones and practical application of cloning techniques. For example, abnormal development of cloned fetuses, anatomical abnormality of cloned offspring, postpartum mortality and low production efficiency of healthy clones are problems commonly encountered with all animal species [2][3][4].Somatic cell cloning comprises replication of an individual animal. It is of considerable biological significance to ascertain the capability of SCNT technology in producing multiple generations of clones, namely, sequential cloning of cloned animals. To date, mice have been cloned up to six generations [5], while cows have been cloned up to two generations [6].Serial cloning of pigs has practical implications in producing genetically engineered animals. Genetically modified pigs such as alpha 1,3-galactosyltransferase-knockout pigs [7][8][9][10][11][12][13][14] and those with a n-3 fatty acid desaturase gene [15] have been produced using serial cloning technology. In this context, multiple generations of clones up to the third generation have been reported in pigs [16,17]. On the other hand, studies have found that serial cloning causes the accumulation of epigenetic modifications that result from the SCNT procedure [3,18]. Indeed, the production efficiency of clones has been reported to decrease as the number of serial clonings increases [5,6,17].Telomere shortening is another form of genetic modification resulting from the somatic cell cloning procedure [19][20][21][22][23][24]. However, serial somatic cell cloning has been shown to not cause telomere shortening in mice [5] and cows [6]. The changes in telomere length involved in serial pig cloning have yet to be determined....

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“…Alpha-GalT is a key enzyme that mediates synthesis of the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species [16], and removal of the epitope is considered to be a prerequisite for xenotransplantation [17]. Thus, creation of genetically modified pig individuals that lack expression of the α-Gal epitope has been the main target for pig-to-human xenotransplantation, and this can be performed by somatic cell nuclear transfer (SCNT) of porcine cells lacking expression of α-GalT mRNA synthesis by gene targeting [18][19][20][21][22][23][24][25]. However, compared with the RNAi-based approach, this approach is labor intensive and time consuming, and it is sometimes difficult to obtain knockout pigs.…”

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Successful Suppression of Endogenous <i>α-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>

Chi

Shinohara

Yokomine

et al. 2012

Journal of Reproduction and Development

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Abstract. RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B 4 , a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of doublestranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation. Key words: Blastocyst, α-1,3-Galactosyltransferase, Pig, RNAi, Somatic cell nuclear transfer (J. Reprod. Dev. 58: [69][70][71][72][73][74][75][76] 2012) S uppression of endogenous target gene expression by gene targeting has been proven as a powerful tool for exploration of the biological function of a gene of interest. Recently, attempts to use antisense technology, now termed RNA interference (RNAi), as a convenient and valuable tool for understanding the in vivo function of a gene of interest have been made [1][2][3][4][5][6][7][8]. RNAi is a multiple-step process that involves the generation of 21-25 nt small interfering (si) RNA and results in degradation of the homologous RNA [2]. Since siRNA can be easily synthesized chemically, it is easy to study the role of endogenous genes by injecting double-stranded (ds) RNA or siRNA into early embryos. However, in the former cases, the effectiveness of dsRNA introduced into cultured cells is known to be retained for a relatively short time (less than one week) [5][6][7][8][9]. In contrast, use of siRNA should be more beneficial for conferring long-term expression of siRNA in transfected cells. In fact, this technology (which is termed "transgenic RNAi") was found to be effective in knocking down ta...

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α1,3-Galactosyltransferase Gene-Knockout Miniature Swine Produce Natural Cytotoxic Anti-Gal Antibodies

Cited by 81 publications

References 11 publications

Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

Production Efficiency and Telomere Length of the Cloned Pigs Following Serial Somatic Cell Nuclear Transfer

Successful Suppression of Endogenous <i>α-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>

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α1,3-Galactosyltransferase Gene-Knockout Miniature Swine Produce Natural Cytotoxic Anti-Gal Antibodies

Cited by 81 publications

References 11 publications

Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

Production Efficiency and Telomere Length of the Cloned Pigs Following Serial Somatic Cell Nuclear Transfer

Successful Suppression of Endogenous <i>&alpha;-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>

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Successful Suppression of Endogenous <i>α-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>