Development of Embryo Cryopreservation Procedures for Laboratory Rats

实验室大鼠胚胎冷冻保存程序的开发

• 批准号: 6432960
• 负责人: WILLIAM F. RALL
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432960
• 关键词: cryopreservation; disease /disorder model; embryo /fetus cell /tissue; genetic registry /resource /referral center; genetic strain; genotype; gonadotropin releasing factor; laboratory rat; method development; technology /technique development; tissue /cell preparation; tissue resource /registry

The NIH Animal Genetic Resource (NIHAGR) maintains many unique rat genotypes used as models of normal and disease processes in man. Effective management of this resource would be facilitated by the development of embryo cryopreservation procedures for long-term banking. In order to develop banks of cryopreserved germ plasm from laboratory rats, protocols for each step of the embryo banking and rederivation process must be established. These would include the preparation and mating of embryo donor females, embryo collection, cryopreservation, thawing, and embryo transfer. Previous studies suggest that superovulation procedures developed for laboratory mice are not as effective when applied to laboratory rats. The principal objective of this project is to evaluate treatment with an analog of gonadotropin releasing hormone (GnRH-a) as an alternative approach to synchronize estrus cycles for embryo collection and cryopreservation. Our efforts to date have yielded the world's largest bank of cryopreserved rat embryos. A total of approximately 58,000 embryos have been cryopreserved from 109 rat strains and stocks. Seventy rat strains and stocks exist solely as banked embryos. The efficiency of embryo collection varies between models in a manner similar to that of the breeding performance of the model. However, progress has been made in developing banks of cryopreserved embryos from all rat models examined to date. Four recent requests by investigators to reestablish rat models that existed only as frozen embryos demonstrate the usefulness of rat embryo banking. For three of the models, LER/N, LEW/N-op (osteopetrosis), and LEW/N-tl (toothless), the foundation colonies were disbanded after, respectively, 75, 128, and 179 embryos were banked. In the fourth case, M520/N-lr (leaner), a "minimal bank" of 367 embryos was established before the foundation colony was disbanded. Nevertheless, sufficient numbers of pups were obtained after thawing and transfer of, respectively, 23, 94, 55, and 76 embryos to recover breeders to reestablish foundation colonies. In each case, three of the criteria for establishing a bank have been met: pups were rederived from banked rat embryos, rederived pups exhibited the desired genotype, and the rederived pups have bred and produced pups. Embryo banking of these four models to reach our final criteria, 500 banked embryos, is nearly complete.

美国国立卫生研究院动物遗传资源 (NIHAGR) 保留了许多独特的大鼠基因型，用作人类正常和疾病过程的模型。 开发用于长期储存的胚胎冷冻保存程序将有助于对该资源的有效管理。 为了开发实验室大鼠的冷冻种质库，必须建立胚胎库和再衍生过程每个步骤的方案。这些包括胚胎供体雌性的准备和交配、胚胎采集、冷冻保存、解冻和胚胎移植。 先前的研究表明，为实验室小鼠开发的超排卵程序应用于实验室大鼠时并不那么有效。 该项目的主要目标是评估促性腺激素释放激素 (GnRH-a) 类似物的治疗作为同步胚胎采集和冷冻保存发情周期的替代方法。迄今为止，我们的努力已经产生了世界上最大的冷冻大鼠胚胎库。总共冷冻保存了 109 种大鼠品系和种群的约 58,000 个胚胎。 七十种大鼠品系和种群仅以储存胚胎的形式存在。胚胎收集效率在模型之间存在差异，其方式类似于模型的育种性能。然而，迄今为止，在开发所有大鼠模型的冷冻胚胎库方面已经取得了进展。研究人员最近提出四项要求，要求重建仅以冷冻胚胎形式存在的大鼠模型，这证明了大鼠胚胎库的有用性。对于 LER/N、LEW/N-op（骨质疏松）和 LEW/N-tl（无牙）这三种模型，基础集落分别在 75、128 和 179 个胚胎入库后解散。在第四个案例中，M520/N-lr（更精简），在基础群体解散之前建立了一个包含 367 个胚胎的“最小库”。尽管如此，在解冻和移植分别23、94、55和76个胚胎后获得了足够数量的幼崽，以恢复繁殖者以重建基础群体。在每种情况下，都满足了建立银行的三个标准：从储存的大鼠胚胎中再衍生出幼仔，再衍生的幼仔表现出所需的基因型，并且再衍生的幼仔已经繁殖并产生了幼仔。这四种模型的胚胎库已接近完成，以达到我们的最终标准（500 个胚胎库）。