Safeguarding Genetic Resources of Aquatic Biomedical Models - Research Supplemental

项目摘要/摘要： 在水生生物医学模型中，创建和表征了数千条有价值的研究线路 每年通过各种方法，包括诱变，基因转移，基因编辑，基因敲除， 杂交和回交。在NIH资助的五个水产养殖中心中，单倍体的保存 种质(精子)可用于三个种群：斑马鱼、剑鱼和非洲爪哇。目前有 目前还没有实用的方法来保存二倍体种质(如胚胎或幼虫) 水族。这个问题对于海兔来说尤其突出，因为 生殖性状(非自交两性)需要早期生命阶段的超低温保存。 而不是精子。超低温保存的二倍体种质资源的可获得性将大大加快 通过消除对筛选和生产多种产品的需求来获得大量研究生产线 世世代代产生纯合子。因此，社会需要保存和利用基因 所有水生生物医学模型的资源将通过新技术得到极大的提升 胚胎和幼虫的冷冻保存，这项技术对于海兔来说是必不可少的。因此，我们的目标是 开发新的电低温生物学技术以监测和改进玻璃化和冷冻回收 水生生物医学模型的二倍体基因组方案。具体目标是：1)发展小说 与海兔相关的低温过程的超快速监测技术(毫秒级) 胚胎玻璃化冷冻；2)使用新的监测技术，获得对传统 海兔胚胎玻璃化冷冻的方法，以及3)扩展监测技术以允许相互作用 利用低温技术获得海兔胚胎玻璃化冷冻的新方法。这项工作，尽管 侧重于海兔作为补充剂，将对青蛙、火蜥蜴的胚胎和精子有用， 和鱼类，并将提供未来拨款提案所需的初步数据，以扩大用途和 申请。这一补充将是非常有价值的推进家长奖，通过添加 强大的新方法(目标1)，将极大地加速我们当前的常规研究(AIM 2)，它将为我们提供建立新方法的新工具(目标3)。家长奖(和 其他R24资金)使我们能够组建一个独特的跨学科小组，拥有超过15年的经验 这项工作的所有方面都有专业知识，包括生殖生物学、低温生物学、工程学和设备 捏造。这项工作可以在本项目年度(进入第三年)内完成，但不能 与父母奖励或先前补充资金资助的工作重叠。

Project Summary/Abstract: Within aquatic biomedical models, thousands of valuable research lines are created and characterized each year by numerous methods including mutagenesis, gene transfer, gene editing, gene knockout, hybridization, and backcrossing. Among the five NIH‐funded aquatic stock centers, preservation of haploid germplasm (sperm) is possible for three groups: zebrafish, Xiphophorus and Xenopus. Currently there is no practical method available for preservation of diploid germplasm (e.g., embryos or larvae) for any aquatic group. This problem is especially prominent for the sea hare, Aplysia californica, which because of reproductive traits (non‐self‐fertilizing hermaphroditism) requires cryopreservation of early life stages rather than sperm. The availability of cryopreserved diploid germplasm would greatly accelerate the availability of numerous research lines by removing needs for screening and production of multiple generations to produce homozygosity. Thus, the community needs for preservation and use of genetic resources would be greatly advanced for all aquatic biomedical models by novel technologies for cryopreservation of embryos and larvae, and this technology is essential for Aplysia. Therefore, our goal is to develop novel electro‐cryobiology technologies to monitor and improve vitrification and cryo‐recovery protocols for diploid genomes of aquatic biomedical models. The Specific Aims are to: 1) develop novel technologies for ultra‐rapid monitoring (millisecond) of cryogenic processes associated with Aplysia embryo vitrification; 2) use the novel monitoring technology to gain new insights into traditional approaches for Aplysia embryo vitrification, and 3) expand the monitoring technology to allow interaction with cryogenic processes to gain new approaches for Aplysia embryo vitrification. This work, although focused on Aplysia for the supplement, would be useful for embryos and sperm of frogs, salamanders, and fishes, and would provide preliminary data needed for future grant proposals to expand utility and application. This supplement would be extremely valuable in advancing the parent award by adding powerful new approaches (Aim 1) that would greatly accelerate our current conventional research (Aim 2), and it would provide us with novel tools to establish new approaches (Aim 3). The parent award (and other R24 funding) has allowed us to assemble a unique interdisciplinary group with more than 15 years of expertise in all aspects of this work spanning reproductive biology, cryobiology, engineering, and device fabrication. This work can be completed within the current project year (entering Year 3), and it does not overlap with work funded in the parent award or through previous supplemental funding.