Development of cloned pig embryos by nuclear transfer following different activation treatments

Kim, Yang-Sil; Lee, Sung‐Lim; Ock, Sun‐A; Balasubramanian, S.; Choe, Sang-Yong; Rho, Gyu‐Jin

doi:10.1002/mrd.20211

Cited by 29 publications

(25 citation statements)

References 47 publications

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“…Our research, as well as that of others, has demonstrated that chromatin remodeling and blastomere ploidy frequently deviate from normal after NT in various species, including cattle (Booth et al 2003, Bureau et al 2003, Li et al 2004a, 2004b, rabbits (Shi et al 2004) and pigs (Kim et al 2005). Several significant morphologic changes occur in the donor nucleus after NT into cytoplasts with high MPF activity.…”

Section: Discussionsupporting

confidence: 67%

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Aston¹,

Li²,

Hicks³

et al. 2006

Reproduction

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This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stage in vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.

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Section: Discussionsupporting

confidence: 67%

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Aston¹,

Li²,

Hicks³

et al. 2006

Reproduction

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“…Moreover, the rates of cleavage and blastocyst formation of parthenotes treated with LatA were significantly higher than in the control. Therefore, it is suggested that the LatA treatment was effective in inhibiting extrusion of a pPB in SCNT embryos after artificial activation.The loss of chromosomes by pPB formation is thought to be the cause of developmental failure of SCNT embryos, and in porcine SCNT technology, CB has been widely used to prevent extrusion of a pPB, which contains half of the chromosomes of a donor cell at postactivation, reducing the aneuploidy leading to false development of SCNT embryos [15,31,32]. In the present study, the rate of blastocyst formation of SCNT embryos treated with LatA was significantly higher than that of the control.…”

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confidence: 42%

Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

Himaki

Mizobe

Tsuda

et al. 2012

Journal of Reproduction and Development

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Abstract. The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term. Key words: Aged donor, Clawn miniature pig, Cytoskeletal inhibitors, Latrunculin A, Nuclear transfer (J. Reprod. Dev. 58: [398][399][400][401][402][403] 2012) C lawn miniature pigs (inbred strain) have a potential value for biomedical research and xenotransplantation due to their clear genetic background [1][2][3] and few individual differences; thus, they have stimulated investigation of novel strategies directed at generating a genetically modified miniature pig by somatic cell nuclear transfer (SCNT). We have shown how to produce live offspring derived from Clawn miniature pig SCNT embryos; however, the efficiency still remains low [4]. To date, pigs have been cloned with SCNT using donor cells derived from fetal pigs to pigs several years of age. The efficiency of SCNT is influenced by many factors, including quality of recipient oocytes, type of donor cells, reprogramming of donor nuclei and condition of recipient gilts [4][5][6][7][8][9][10][11][12][13]. In particular, the diploid complement retention is one of the most important factors in improving the developmental ability of SCNT embryos [14,15]. SCNT embryos derived from diploid cells are generally treated with cytoskeletal inhibitors, such as cytochalasin B (CB), after activation to prevent extrusion of a pseudo polar body (pPB) and the possible loss of chromosomes maintain normal ploidy [7,16,17]. Several reports have shown that CB treatment of SCNT embryos resulted in increased blastocyst formation in vitro [18][19][20][21]. However, it has been reported that postactivation treatment with CB had no effect on either blastocyst rate or cell number of SCNT embryos [9,22]. CB possess cytotoxicity [23], and its cytotoxicity might have a detrimental effect on the subsequent development of SCNT embryos.Latrunculin A (LatA), a toxin purified from the Red Sea sponge Latrunculia magnifica, is a member of the cytoskel...

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“…The NT procedure was performed according to the previously described protocol [11] with minor modifications. Briefly, ovaries of domestic pigs were obtained from prepubertal gilts at a local slaughterhouse.…”

Section: Nuclear Transfer (Nt)mentioning

confidence: 99%

Developmental Ability of Miniature Pig Embryos Cloned with Mesenchymal Stem Cells

Lee

Kang

Maeng

et al. 2010

Journal of Reproduction and Development

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Abstract. The present study compared the developmental ability of miniature pig embryos cloned with fetal fibroblasts (FFs), bone marrow-derived mesenchymal stem cells (MSCs) and differentiated (osteocytes, adipocytes and chondrocytes) MSCs. MSCs were isolated from an approximately 1-month-old female miniature pig (T-type, PWG Micro-pig ® , PWG Genetics Korea). MSCs were differentiated into osteocytes, adipocytes and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, as well as FFs of miniature pig, were transferred into enucleated oocytes of domestic pigs. Data from 10 replicates involving 1567 cloned embryos were assessed in terms of developmental rates. The in vitro development rate to the blastocyst stage of embryos cloned with undifferentiated MSCs was significantly (P<0.05) higher than that of embryos cloned with differentiated MSCs and FFs. Surgical transfer of 523 two-cell stage embryos cloned with undifferentiated MSCs into five synchronized domestic pig recipients resulted in 5 cloned miniature pig offspring (1 stillborn and 4 viable) from 2 pregnant recipients. The results imply that MSCs might be multipotent and that they can be used to produce viable cloned miniature pigs that cannot be easily reproduced with differentiated somatic cells. Key words: Animal cloning, Differentiation, Mesenchymal stem cells, Miniature pig, Nuclear transfer (J. Reprod. Dev. 56: [256][257][258][259][260][261][262] 2010) he production of cloned animals by nuclear transfer (NT) has been increasingly studied as a potential approach to tissue and organ xenotransplantation with reduced immunogenicity for endstage organ failure. Several studies have suggested that miniature pigs, rather than nonhuman-primate species, are suitable as a donor animal for human xenotransplantation [1,2] because miniature pigs are superior in terms of similarities in gross anatomy and physiology to humans as well as in terms of ethical issues. The NT efficiency, however, remains relatively low due to abnormalities throughout pre-and post-implantation development regardless of the species or type of donor cell [3]. A number of factors affect NT efficiency, including the NT technical process, activation protocol, recipient oocyte age, cell cycle stage and type of donor cells, which has been implicated in the precise reprogramming of chromatin and genomic imprinting. Among these factors, incomplete selection of donor cells has also been considered a prime cause for NT inefficiency.Transfer of embryonic stem (ES) cells to produce cloned embryos has resulted in consistently higher numbers of viable offspring compared with somatic cells in mice [4,5]. Moreover, embryos cloned with porcine mesenchymal stem cells (MSCs) and their derivatives along the osteogenic lineage give rise to an increase in the rate of preimplantation development compared with adult fibroblasts [6]. Our previous study on gene expression profiles demonstrated that some ge...

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Development of cloned pig embryos by nuclear transfer following different activation treatments

Cited by 29 publications

References 47 publications

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

Developmental Ability of Miniature Pig Embryos Cloned with Mesenchymal Stem Cells

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