Timing and Uniformity of Embryonic Gene Activation Affect Subsequent Pre-implantation Development of Cloned Bovine Embryos

Kasamatsu, A.; Saeki, Kazuhiro; Tamari, Tomohiro; Iwamoto, D.; Tatemizo, Atsuhiro; Matsumoto, Kazuya; Hosoi, Yoshihiko; Iritani, Akira

doi:10.1262/jrd.19005

Cited by 7 publications

(5 citation statements)

References 34 publications

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“…This effect was also suggested by the earlier expression of egfp in chemically activated porcine, bovine, feline and equine embryos in the present study. In addition, earlier expression of endogenous and exogenous genes was observed for parthenogenetic and cloned embryos assisted by chemical activation (Wrenzycki et al 2006;Kasamatsu et al 2007). The timing of egfp expression in non-activated embryos was not possible to determine because preliminary experiments indicated developmental arrest before the morula stage after ICSI without chemical activation in porcine, bovine and feline embryos (data not shown).…”

Section: Discussionmentioning

confidence: 99%

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

Pereyra-Bonnet

Fernández-Martı́n

Olivera

et al. 2008

Reprod. Fertil. Dev.

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Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.

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Section: Discussionmentioning

confidence: 99%

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

Pereyra-Bonnet

Fernández-Martı́n

Olivera

et al. 2008

Reprod. Fertil. Dev.

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“…Fibroblasts taken from bovine ear tissue were used as controls. The SCNT embryos were then cultured for 168 hours [17]. The SCNT experiments were repeated 3 times.…”

Section: Resultsmentioning

confidence: 99%

“…SCNT by electrofusion of somatic cells with enucleated bovine oocytes was performed as described earlier [17]. The SCNT embryos were activated with 5 µM ionomycin for 5 min and then treated with 10 µg/ml cycloheximide in modified synthetic oviduct fluid medium (mSOFM) without KH 2 PO 4 until 6 h post fusion (hpf) at 39°C in 5% CO 2 , 5% O 2 and 90% N 2 with high humidity.…”

Section: Methodsmentioning

confidence: 99%

“…The SCNT embryos were activated with 5 µM ionomycin for 5 min and then treated with 10 µg/ml cycloheximide in modified synthetic oviduct fluid medium (mSOFM) without KH 2 PO 4 until 6 h post fusion (hpf) at 39°C in 5% CO 2 , 5% O 2 and 90% N 2 with high humidity. Following activation, the SCNT embryos were cultured in mSOFM until 168 hpf [17].…”

Section: Methodsmentioning

confidence: 99%

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Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade

et al. 2009

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Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

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“…After IVF, presumptive zygotes were freed from cumulus cells by pipetting and cultured in a modified synthetic oviduct fluid medium with amino acids (Takahashi & First, 1992) and further modified by excluding KH 2 PO 4 (Kasamatsu et al ., 2007) until 168 hours postinsemination (hpi) at 39 °C under 5% CO 2 , 5% O 2 and 90% N 2 with high humidity. Cleavage and blastocyst development were assessed at 48 and 168 hpi, respectively.…”

Section: Methodsmentioning

confidence: 99%

Enhancement of histone acetylation by trichostatin A duringin vitrofertilization of bovine oocytes affects cell number of the inner cell mass of the resulting blastocysts

Ikeda

Tatemizo

Iwamoto

et al. 2009

Zygote

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Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization to establish a totipotent state for normal development. In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were matured in vitro and subjected to IVF in a defined medium supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF) medium until 168 h postinsemination (hpi). Some oocytes were immunostained using antibody specific for histone H4-acetylated lysine 5 at 10 hpi. Cleavage, blastocyst development and cell number of inner cell mass (ICM) and trophectoderm (TE) of blastocysts were assessed. TSA treatment enhanced histone acetylation that was prominent in decondensed sperm nuclei. TSA did not affect the postfertilization cleavage, blastocyst rates, and TE cell number. However, it significantly enhanced ICM cell number (p < 0.05). These results indicate that TSA treatment during IVF of bovine oocytes does not affect blastocyst development but alters the cell number of ICM, suggesting that overriding epigenetic modification of the genome during fertilization has a carryover effect on cell proliferation and differentiation in preimplantation embryos. Thus, further environmental quality controls in assisted reproductive technologies are needed in terms of factors which affect chromatin remodelling.

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Timing and Uniformity of Embryonic Gene Activation Affect Subsequent Pre-implantation Development of Cloned Bovine Embryos

Cited by 7 publications

References 34 publications

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade

Enhancement of histone acetylation by trichostatin A duringin vitrofertilization of bovine oocytes affects cell number of the inner cell mass of the resulting blastocysts

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