Cryopreservation of pronuclear-stage rabbit zygotes, applicable for production of transgenic rabbits

原核期兔受精卵的冷冻保存，适用于转基因兔的生产

• 批准号: 13660282
• 负责人: HOCHI Shinichi
• 金额: $ 0.64万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660282/
• 关键词: Transgenic rabbits; Pronuclear zygotes; Cryopreservation; Vitrification; Foreign DNA; Human growth hormone; Gel-loading tip; Cryotop

The objective of this study was to develop cryopreservation method of pronuclear-stage rabbit zygotes. Conventional 2-step freezing of zygotes in the presence of 1.5M ethylene glycol + 0.1M sucrose resulted in morphological survival and blastocyst developmental rates of 74 and 52%, respectively. Only 4% (18/414) of the frozen-thawed zygotes followed by DNA microinjection developed into normal offspring, but to our best knowledge they were the first rabbit offspring derived from cryopreserved pronuclear zygotes (Hochi et al., Mol. Reprod. Dev., 2001). Next, some ultra-rapid cooling procedures, that have been reported to be effective in cryopreservation of bovine oocytes, were applied for the pro nuclear-stage rabbit zygotes. When the zygotes were cryopreserved using both Cryotop as a device and 2.7M ethylene glycol + 2.3M DMSO + 0.5M sucrose as CPAs, the maximum morphological survival rate of 100% and blastocyst developmental rate of 51% were obtained. The vitrified-warmed zygotes resulted in production of offspring at 36% per transfer, while fresh control zygotes developed to offspring at the rate of 53% (Hochi et al., Theriogenology, 2003). The Cryotop vitrification method established here for pronuclear-stage rabbit zygotes (in vivo survival 36%) is obviously of practical importance in systematic production of transgenic rabbits by pronuclear microiniection of foreign DNA.

本研究旨在建立原核期兔受精卵的冷冻保存方法。在1.5M乙二醇+0.1M蔗糖的存在下，常规的两步冷冻受精卵的形态存活率和囊胚发育率分别为74%和52%。只有4%（18/414）的冻融受精卵随后进行DNA显微注射，发育成正常后代，但据我们所知，它们是第一批来自冻存原核受精卵的兔后代（Hochi et al.，摩尔Reprod.偏差：2001年）的报告。接下来，一些超快速冷却程序，已被报道是有效的牛卵母细胞冷冻保存，适用于原核阶段的兔受精卵。当以Cryotop为冷冻装置，以2.7M乙二醇+2.3M DMSO +0.5M蔗糖为冷冻保护剂时，受精卵的形态存活率最高，为100%，囊胚发育率最高，为51%。玻璃化加温的受精卵导致每次转移产生36%的后代，而新鲜对照受精卵以53%的比率发育成后代（Hochi等人，Theriogenology，2003）。本文建立的原核期兔受精卵的Cryotop玻璃化冷冻方法（体内存活率为36%），对原核显微注射外源DNA系统地生产转基因兔具有重要的实用价值。

项目成果

Shinichi Hochi 他: "Effects of Cryopreservation of Pronuclear-Stage Rabbit Zygotes on the Morphological Survival, Blastocyst Formation, and Full-Term Development After DNA Microinjection"Molecular Reproduction and Development. 60・2. 227-232 (2001)

Shinichi Hochi 等人：“原核阶段兔受精卵冷冻保存对 DNA 显微注射后形态存活、囊胚形成和足月发育的影响”，分子复制与发育 60・2。

Shinichi HOCHI et al.: "Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure"Theriogenology. 60(Accepted). (2003)

Shinichi HOCHI 等人：“通过最小体积冷却程序成功玻璃化原核阶段兔受精卵”动物发生学。

S. Hochi et al.: "Effects of cryopreservation of pronuclear-stage rabbit zygotes on morphological survival, blastocyst formation, and full-term development after DNA microinjection"Mol. Reprod. Dev.. 60-2. 227-232 (2001)

S. Hochi 等人：“原核阶段兔受精卵冷冻保存对 DNA 显微注射后形态存活、囊胚形成和足月发育的影响”Mol。

Sinichi Hochi 他: "Vitrification of pronuclear-stage rabbit zygotes by different ultra-rapid cooling procedures"Theriogenology. 59・1. 303 (2003)

Sinichi Hochi 等人：“通过不同的超快速冷却程序对原核阶段兔受精卵进行玻璃化”，Theriogenology 59・1（2003）。

Shinichi HOCHI et al.: "Effects of cryopreservation of pronuclear-stage rabbit zygotes on morphological survival, blastocyst formation, and full-term development after DNA-microinjection"Molecular Reproduction and Development. 60. 227-232 (2001)

Shinichi HOCHI 等人：“原核阶段兔受精卵冷冻保存对 DNA 显微注射后形态存活、囊胚形成和足月发育的影响”《分子生殖与发育》。