Production of transgenic mice by microinjection of DNA into vitrified pronucleate stage eggs

Tada, Norio; Kasai, Kazuhiro; Ogawa, Shyoso

doi:10.1007/bf01968786

Cited by 20 publications

(15 citation statements)

References 22 publications

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“…If an inventory of cryopreserved embryos is available, the scheduling limitation could be eliminated and production rate can be largely increased. Cryopreserved zygotes have been successfully proven to be fit for transgenesis via DNA microinjection in mouse by both slow cooling (Leibo et al, 1991;Keskintepe et al, 2001) and vitrification (Tada et al, 1995;Bagis et al, 2002).…”

Section: Applications Of Embryo Cryopreservation Mouse Colony Managemmentioning

confidence: 99%

Cryopreservation of mammalian embryos: Advancement of putting life on hold

Tsang

Chow

2010

Birth Defects Research Pt C

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Rodent transgenesis and human-assisted reproductive programs involve multistep handling of preimplantation embryos. The efficacy of production and quality of results from conventionally scheduled programs are limited by temporal constraints other than the quality and quantities of embryos per se. The emergence of vitrification, a water ice-free cryopreservation technique, as a reliable way to arrest further growth of preimplantation embryos, provides an option to eliminate the time constraint. In this article, current and potential applications of cryopreservation to facilitate laboratory animal experiments, colony management, and human-assisted reproductive programs are reviewed. Carrier devices developed for vitrification in the last two decades are compared with an emphasis on their physical properties that infer cooling rate of samples and sterility assurance. Biological impacts of improved cryopreservation on preimplantation embryos are also discussed.

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Section: Applications Of Embryo Cryopreservation Mouse Colony Managemmentioning

confidence: 99%

Cryopreservation of mammalian embryos: Advancement of putting life on hold

Tsang

Chow

2010

Birth Defects Research Pt C

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“…The vitrification in this study was performed by the method of Tada et al [27,28] and Bagis et al. [29], with slight modifications.…”

Section: Cooling and Warming Of Embryosmentioning

confidence: 99%

“…The vitrification in this study was performed by the method of Tada et al [27,28] and Bagis et al [29], with slight modifications. Embryos were suspended in an equilibration solution consisting of 4% (v/v) EG at 37 C for 15 min.…”

Section: Cooling and Warming Of Embryosmentioning

confidence: 99%

Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mouse Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mouse Embryos

Kim

Yong

Lee

et al. 2008

Journal of Reproduction and Development

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Abstract. In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on in vitro and in vivo development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 μl of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO2 in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For in vivo evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 vs. 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos. Key words: Cryopreservation, Embryo, Mouse, Polyvinylpyrrolidone (PVP), Vitrification (J. Reprod. Dev. 54: [250][251][252][253] 2008) ince the first report of mouse embryo cryopreservation was published in 1972 [1,2], there have been many technical improvements in cryoprotectant (CPAs) preparation. Factors known to affect the cryopreservation of mouse embryos involve the effectiveness of different CPAs, equilibration times of CPAs, cooling and warming rates, osmotic pressures of CPAs, temperatures for various cooling conditions and dilution process [3][4][5][6][7][8][9].The use of vitrification protocols is a way to avoid cell damage caused by intracellular ice formation, osmotic and chilling injury and zona and blastomere fracture. Vitrification as an ultrarapid cooling technique has been used in cryopreservation of cells, tissues and organs at low temperatures by creating a completely vitreous (glass-like) state [10]. At a sufficiently low temperature, cooling solutions become highly viscous, and solidification occurs without ice formation. Ice formation is usually observed when the CPA concentration is higher than 40% (w/v) [11]. Improvements in ice formation have been made 1) by selecting the most appropriate CPAs, 2) by using a mixture of two or more CPAs, macromolecules and nonpermeating agents and 3) by using stepwise equilibration (two or three steps) in vitrification solutions of intermediate concentration at room temperature or after ...

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“…The present limited use of transgenic technology in rabbits, however, might be due to the requirement of larger animal facilities and the necessity to have zygotes readily available for DNA microinjection. More ef®cient availability of cryopreserved pronuclear-stage rabbit zygotes for DNA microinjection, as already reported in mice (Leibo et al, 1991;Tada et al, 1995) and rats (Takahashi et al, 1999) might encourage the use of rabbits for transgenic studies.…”

Section: Introductionmentioning

confidence: 85%

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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Production of transgenic mice by microinjection of DNA into vitrified pronucleate stage eggs

Cited by 20 publications

References 22 publications

Cryopreservation of mammalian embryos: Advancement of putting life on hold

Cryopreservation of mammalian embryos: Advancement of putting life on hold

Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mouse Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mouse Embryos

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

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