Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage

Ito, Kazumi; Otake, S; Hirabayashi, Masumi; Hochi, Shinichi; Ueda, Masatsugu

doi:10.1016/s0093-691x(98)00210-6

Cited by 11 publications

(7 citation statements)

References 27 publications

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“…But in the present study, only 13.4% of 388 fresh zygotes that were transferred after DNA microinjection developed into live young, suggesting that the microinjected DNA adversely affected fetal development. The developmental potential of DNA-injected zygotes has been reported to be considerably reduced in mice , rats (Hirabayashi et al, 1997), rabbits (Chrenek et al, 1998), and cattle (Peura et al, 1995;Ito et al, 1998). We also observed even further reduction in the rate of in vivo development of cryopreserved rabbit zygotes after DNA injection, indicating a synergistic deleterious effect of microinjection and cryopreservation (Tables 2 and 3).…”

Section: Discussionsupporting

confidence: 55%

Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

Hochi

Hirabayashi²,

Hirao³

et al. 2001

Molecular Reproduction Devel

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The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

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Section: Discussionsupporting

confidence: 55%

Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

Hochi

Hirabayashi²,

Hirao³

et al. 2001

Molecular Reproduction Devel

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“…In our experiment, the negative effect of transgene introduction occurred in the fi rst cell cycle because the microinjected embryos developed better (61.1%) from cleavage to the blastocyst stage than the control embryos (48.6%). This negative effect on zygote development may result from adverse infl uence of the high forces used for visualization of the pronuclei and opening of the plasma membrane during micromanipulation (Ito et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

Obtaining calves after transfer of embryos microinjected with the human interferon alpha (IFNα) gene, pbLGIFN-GFPBsd

Duszewska¹,

Lipiński²,

Gawron³

et al. 2008

J. Anim. Feed Sci.

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The objective of this study was to obtain calves with an integrated human interferon alpha (IFNα) gene. The transgene (pbLGIFN-GFPBsd) was introduced by microinjection into one of the zygote's pronuclei. The transgene (pbLGIFN-GFPBsd) contained the human interferon alpha (IFNα) gene, bovine lactoglobulin promoter (bLG), gfp (green fl uorescence protein) and blasticidin resistance gene (bsd). After microinjection of 2107 zygotes, the proportion developing to the blastocyst stage (10.4%) was lower than for non-injected embryos (39.9%; P<0.01). Of the 2107 that were microinjected, 76 were GFP-positive blastocysts (3.6%). Fifty-seven GFP-positive blastocysts and 31 blastocysts from non-injected embryos were transferred singly to recipients. A lower rate of calving was observed after transfer of GFP-positive blastocysts (19.3%) as compared with noninjected embryos (48.4%; P<0.05). All newborn calves were healthy and of normal weight. PCR analyses indicated that none of the calves obtained after transfer of GFP-positive blastocysts carried the human interferon alpha gene.

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“…Presumptive zygotes were stripped of cumulus cells and then cultured in modified synthetic oviductal fluid (m-SOF) [13] at 39 C in 5% CO2, 5% O2 and 90% N2. Mid to expanded blastocysts, which were constituted from 119.6 ± 31.6 cells (mean ± SD) [14], were harvested on Day 7 (the day of IVF = Day 0).…”

Section: In Vitro Production Of Bovine Blastocystsmentioning

confidence: 99%

Effect of Time Interval between Biopsy and Vitrification on Survival of In Vitro-Produced Bovine Blastocysts.

Ito¹,

Sekimoto

Hirabayashi³

et al. 1999

Journal of Reproduction and Development

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Abstract. The aim of this study was to improve the cryosurvival of bovine blastocysts following biopsy. In vitro-derived bovine blastocysts were biopsied with a metal blade, then cultured for 0 to 20 h in m-SOF and then vitrified in the presence of ethylene glycol and dimethyl sulfoxide. The post-warm survival rates after 24 h of in vitro culturing of the biopsied embryos were significantly higher at 1.25, 2.5, 5 and 10 h than that of 0 and 20 h (69, 73, 78 and 64% vs 34 and 27%, p<0.05, respectively). The survival rate of embryos vitrified at 2.5 and 5 h after biopsy was not different from that of the non-vitrified control group (73 and 78% vs 96%, p=0.08 vs 0.07, respectively). These results indicate that 1.25 to 10 h culture between biopsy and vitrification improves the viability of bovine blastocysts produced in vitro.

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Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage

Cited by 11 publications

References 27 publications

Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

Obtaining calves after transfer of embryos microinjected with the human interferon alpha (IFNα) gene, pbLGIFN-GFPBsd

Effect of Time Interval between Biopsy and Vitrification on Survival of In Vitro-Produced Bovine Blastocysts.

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