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; Sato, Masahiro; Takao, Sonsin; Yoshida, Mitsutoshi

doi:10.1093/biolreprod/81.s1.204

Cited by 3 publications

(7 citation statements)

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“…(5) Improve the ability of the chromosomes to segregate properly. Chromosome segregation may be enhanced by culturing with actin polymerization inhibitors compounds such as latrunculin A that have been shown to improve SCNT pig embryo development when used after cell fusion and activation (Himaki et al, 2010). Cell‐cycle synchronization may be important as it is thought that donor cells in G1 or G0 of the cell cycle would be the most likely to undergo normal replication (Campbell et al, 1993; Prather, 1996).…”

Section: Methods To Improve Nuclear Remodeling and Reprogrammingmentioning

confidence: 99%

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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Section: Methods To Improve Nuclear Remodeling and Reprogrammingmentioning

confidence: 99%

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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“…SCNT miniature pig embryos were produced as described previously [ 22 ] and activated by ultrasound stimulation [ 23 ]. Briefly, oocytes with a polar body were treated with 0.5 μg/ml demecolcine (Sigma-Aldrich Japan (Tokyo, Japan) and 20 mM sucrose for 0.5–1 h. Enucleation was performed by aspirating the first polar body and protruding membrane using a 15 μm inner diameter glass pipette.…”

Section: Methodsmentioning

confidence: 99%

“…After enucleation, a single donor cell was inserted into the perivitelline space of each oocyte using the same glass pipette. Membrane fusion of the cytoplast and donor cell was induced by applying a single direct-current pulse [ 22 ]. Following the fusion pulse, the complexes were cultured for 2 h under 5% CO 2 , 5% O 2 , and 90% N 2 at 38.5°C, in a 100 μl droplet of modified PZM-3 (mPZM-3) [ 22 ] until activation.…”

Section: Methodsmentioning

confidence: 99%

“…Membrane fusion of the cytoplast and donor cell was induced by applying a single direct-current pulse [ 22 ]. Following the fusion pulse, the complexes were cultured for 2 h under 5% CO 2 , 5% O 2 , and 90% N 2 at 38.5°C, in a 100 μl droplet of modified PZM-3 (mPZM-3) [ 22 ] until activation. Fusion status was determined by microscopic examination 1.5 h after applying the pulse.…”

Section: Methodsmentioning

confidence: 99%

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Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma

et al. 2015

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High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)–transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

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“…Latrunculin A, an actin polymerization inhibitor, decreased chromosome segregation errors, and improved the birth rate of cloned embryos (Fig. 2D) [54,55]. However, cloned embryo-specific epigenetic abnormalities such as dimethylation of H3K9me2 were not prevented by latrunculin A.…”

Section: (Ii) Histone Deacetylase Inhibitors Enhance the Activation Omentioning

confidence: 96%

Towards Further Optimization of Preimplantation Embryo Culture Media: from the Viewpoint of Omics and Somatic Cell Nuclear Transfer (SCNT) Studies

Yamada

Hamatani

Akutsu

et al. 2016

Journal of Mammalian Ova Research

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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 3 publications

References 0 publications

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

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

Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma

Towards Further Optimization of Preimplantation Embryo Culture Media: from the Viewpoint of Omics and Somatic Cell Nuclear Transfer (SCNT) Studies

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