Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin

Hinrichs, Katrin; Choi, Young-Ho; Varner, D.D.; Hartman, David L.

doi:10.1530/rep-07-0069

Cited by 56 publications

(47 citation statements)

References 28 publications

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“…large offspring syndrome. The horse appears not to suffer from this syndrome; it has not been reported after transfer of either IVP (Galli et al 2007, Hinrichs et al 2007b or nuclear transfer embryos , Hinrichs et al 2006, 2007a. However, we found in the present study that transfer of IVP equine embryos to the uterus resulted in normalized expression patterns.…”

Section: Pou5f1 In Equine Embryoscontrasting

confidence: 59%

The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts

Choi¹,

Harding²,

Hartman³

et al. 2009

Reproduction

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The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout the blastocysts. In contrast, in vivo-produced day-7 to -10 blastocysts showed greatly reduced trophoectodermal POU5F1 protein expression. To determine whether the uterine environment modulates POU5F1 expression, IVP blastocysts were transferred to the uteri of mares, then recovered 2-3 days later (IVP-ET embryos). These embryos showed similar POU5F1 expression as the in vivo-produced embryos. Levels of POU5F1, SOX2, and NANOG mRNA in IVP-ET blastocysts were significantly higher in the inner cell mass than in trophectoderm (TE) cells. These data suggest that the differentiation of equine TE, as indicated by loss of POU5F1 expression, is impaired during in vitro culture, but proceeds normally when the embryos are exposed to the uterine environment. Previously reported differences in trophectodermal expression of POU5F1 among species may thus be in part artifactual, i.e. related to in vitro culture. Failure for correction of such changes by the uterine environment is a potential factor in the placental abnormalities seen after transfer of cultured embryos in some species.

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Section: Pou5f1 In Equine Embryoscontrasting

confidence: 59%

The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts

Choi¹,

Harding²,

Hartman³

et al. 2009

Reproduction

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“…The cell cycle of the donor cell is crucial for success and G0 or G1 stages have been used in horses. These stages can be achieved through serum starvation, contact inhibition or the use of kinase inhibitors like Roscovitine 25,26 . c) Preparation of enucleated oocytes: After 22-24 h of maturation, the oocytes are discarded of cumulus cells by pipetting 27 .…”

Section: Somatic Cell Nuclear Transfer (Scnt) Methodologymentioning

confidence: 99%

Advances in equine cloning by somatic cell nuclear transfer (SCNT) technique in horses: a review

Wakchaure¹,

Ganguly²

2016

IJB

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Cloning a horse means using the genetic material (DNA) from a donor horse to produce a genetically identical foal. This technique involves collecting the DNA from the donor and inserting that DNA into an egg from another mare whose, DNA content has been removed, fusing donor nucleus with enucleated recipient oocytes, which then develops as an embryo, in vitro culture of embryo and lastly transfer cultured embryo into the uterus of a recipient mare. The modification of the in vitro culture conditions which can be suitable for equine oocyte activation, oocyte maturation and embryo development are the fundamental steps for a successful in vitro procedure for somatic cell nuclear transfer (SCNT) in the horse to avoid the embryo losses. In general, few studies are available in the literature on equine in vitro embryo production and it is only recently that reports have been published on completely in vitro production of equine preimplantation embryos by means of in vitro oocyte maturation. The present review discusses the latest developments in the field of equine cloning technique with the employment of SCNT. The basic understanding of SCNT for in vitro culture conditions is relevant to the increased efficiency of cloning. The available genotype can be used by SCNT which can enhance the vigour of a particular infertile or low fertile animal to produce normal fertility.

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“…For activation, oocytes were placed for 4 min in 5 lM ionomycin in CZB-M without glutamine, nonessential amino acids, or FBS, supplemented with 0.02% polyvinyl alcohol, in air on a warm plate set at 38.2°C. Oocytes were washed and returned to culture in M199E with FBS for 20-30 min, and then were placed in M199HH with 10% FBS and injected with sperm extract as previously described (Hinrichs et al, 2007). After sperm extract injection, oocytes were incubated in the presence of 2 mM 6-dimethylaminopurine (6-DMAP) in embryo culture medium (GB, LifeGlobal, Guilford, CT, USA) with 10% FBS for 4 h in 5% CO 2 in air at 38.2°C, then washed and placed in GB with 10% FBS in an atmosphere of 6% CO 2 , 5% O 2 , and 89% N 2 at 38.2°C.…”

Section: Nuclear Transfermentioning

confidence: 99%

“…S everal articles have been published reporting production of cloned foals (Choi et al, 2009;Choi et al, 2013a;Choi et al, 2014;Galli et al, 2003;Gambini et al, 2012;Hinrichs et al, 2006;Hinrichs et al, 2007;Lagutina et al, 2005). In these reports, the rates of blastocyst production were low, typically < 10%.…”

Section: Introductionmentioning

confidence: 99%

Timing Factors Affecting Blastocyst Development in Equine Somatic Cell Nuclear Transfer

ChoiYoung-Ho

VelezIsabel

Macías-GarcíaBeatriz

et al. 2015

Cellular Reprogramming

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In nuclear transfer (NT), exposure of donor cell chromatin to the ooplast cytoplasm may aid reprogramming; however, the length of exposure feasible is limited by the developmental life span of the oocyte. We examined the effect of duration of nucleus-cytoplasmic exposure before activation and of in vitro maturation (IVM) in equine NT. In experiment 1, 24 h IVM and a delay of 2, 5, or 8 h between reconstruction and activation yielded 4%, 15%, and 11% blastocysts, respectively. In experiment 2, a 5-h activation delay yielded 17% and 22% blastocysts with two donor cell lines. In experiment 3, using a 5-h activation delay, the blastocyst rate was significantly higher using oocytes after 20 h IVM than after 24 h IVM; however, only 28% of oocytes were in metaphase II (MII) at 20 h. In experiment 4, oocytes were denuded of cumulus at 20 h, and those in metaphase I (MI) were returned to culture for 3 h (20+3H treatment); blastocyst rates were 30% and 27%, respectively (8-h and 5-h delay to activation, respectively). Four live foals resulted from the transfer of 17 blastocysts (24%) produced using MII oocytes and a 5- or 8-h activation delay. Use of equine oocytes immediately after reaching MII, combined with a longer delay from reconstruction to activation, increased developmental competence after equine NT.

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Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin

Cited by 56 publications

References 28 publications

The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts

The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts

Advances in equine cloning by somatic cell nuclear transfer (SCNT) technique in horses: a review

Timing Factors Affecting Blastocyst Development in Equine Somatic Cell Nuclear Transfer

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