Latrunculin A Dramatically Improves the Developmental Capacity of Nuclear Transfer Embryos Derived from Gene-Modified Clawn Miniature Pig Cells

Himaki, Takehiro; Mori, Hironori; Mizobe, Yamato; Miyoshi, Katsuhiko; Takao, Sonsin; Yoshida, Mitsutoshi

doi:10.1089/cell.2009.0066

Cited by 13 publications

(10 citation statements)

References 16 publications

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“…In the present study, the rate of blastocyst formation of SCNT embryos treated with LatA was significantly higher than that of the control. This is consistent with a previous study showing that postactivation treatment with LatA was effective in improving the in vitro developmental capacity of cloned miniature pig embryos derived from genetically modified fetal fibroblasts [27]. Moreover, LatA treatment did not appear to have any detrimental effect on the morphology of early SCNT embryos.…”

supporting

confidence: 92%

“…The methods for production of SCNT miniature pig embryos were largely based on those described before [27].…”

Section: Methodsmentioning

confidence: 99%

“…After being washed twice with Hepes-TLP-PVA (Tyrode-lactate-pyruvate-polyvinyl alcohol) and the maturation medium, respectively, COCs with compact cumulus cells and evenly granulated ooplasm were used for the experiments. COCs (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) were cultured in a droplet of maturation medium (200 µl) under paraffin oil (Nacalai Tesque, Kyoto, Japan) in a polystyrene dish (35-mm: Becton Dickinson Labware, Oxnard, CA, USA) and cultured at 38.5 C in an atmosphere of 5% CO 2 in air. The maturation medium was TCM-199 with Earle's salts (Gibco, BRL, Grand Island, NY, USA) supplemented with 0.91 mM sodium pyruvate (Sigma-Aldrich Chemical, St. Louis, MO, USA), 3.05 mM glucose (Wako Pure Chemical Industries, Osaka, Japan), 0.57 mM cysteine hydrochloride hydrate (Sigma), 10 ng/ml epidermal growth factor (Sigma), 10 IU/ml eCG (ASKA Pharmaceutical, Tokyo, Japan), 10 IU/ml hCG (ASKA), 100 µg/ml amikacin sulfate (Meiji Seika, Tokyo, Japan) and 10% (v/v) pig follicular fluid.…”

Section: In Vitro Maturation Of Oocytesmentioning

confidence: 99%

“…After treatment of in vitro matured and denuded oocytes with 0.5 μg/ml demecolcine (Sigma) and 0.02 M sucrose for 0.5-1 h [27], the cytoplasmic protrusion and first PB were removed by aspiration with a 15-μm inner diameter glass pipette. After enucleation, a single donor cell was inserted into the perivitelline space of each oocyte using the same glass pipette.…”

Section: Nuclear Transfermentioning

confidence: 99%

“…LatA can bind specifically to G-actin and has no activity to block the barbed end of F-actin [25]. Recently, we have shown that postactivation treatment with LatA was effective in improving the developmental capacity of cloned miniature pig embryos derived from genetically modified fetal fibroblasts [27]. However, the efficacy of LatA treatment in other donor cell types and the ability of SCNT miniature pig embryos treated with LatA to develop into piglets remain unknown.…”

mentioning

confidence: 99%

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Effect of Postactivation Treatment with Latrunculin A on In Vitro and In Vivo 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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supporting

confidence: 92%

“…The methods for production of SCNT miniature pig embryos were largely based on those described before [27].…”

Section: Methodsmentioning

confidence: 99%

Section: In Vitro Maturation Of Oocytesmentioning

confidence: 99%

Section: Nuclear Transfermentioning

confidence: 99%

mentioning

confidence: 99%

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Effect of Postactivation Treatment with Latrunculin A on In Vitro and In Vivo 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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Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming?

Whitworth

Prather

2010

Molecular Reproduction Devel

100

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SUMMARY Fertile offspring from somatic cell nuclear transfer (SCNT) is the goal of most cloning laboratories. For this process to be successful, a number of events must occur correctly. First the donor nucleus must be in a state that is amenable to remodeling and subsequent genomic reprogramming. The nucleus must be introduced into an oocyte cytoplasm that is capable of facilitating the nuclear remodeling. The oocyte must then be adequately stimulated to initiate development. Finally the resulting embryo must be cultured in an environment that is compatible with the development of that particular embryo. Much has been learned about the incredible changes that occur to a nucleus after it is placed in the cytoplasm of an oocyte. While we think that we are gaining an understanding of the reorganization that occurs to proteins in the donor nucleus, the process of cloning is still very inefficient. Below we will introduce the procedures for SCNT, discuss nuclear remodeling and reprogramming, and review techniques that may improve reprogramming. Finally we will briefly touch on other aspects of SCNT that may improve the development of cloned embryos.

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Pig Cloning Using Somatic Cell Nuclear Transfer

Ouyang

Han

Huang

2020

Methods in Molecular Biology

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Latrunculin A Dramatically Improves the Developmental Capacity of Nuclear Transfer Embryos Derived from Gene-Modified Clawn Miniature Pig Cells

Cited by 13 publications

References 16 publications

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

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

Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming?

Pig Cloning Using Somatic Cell Nuclear Transfer

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