Project

摘要(应用研究) 随着大鼠的出现，新的转基因大鼠模型的产生速度大大加快 胚胎干细胞和基于核酸酶的基因组编辑技术的出现，如ZFN，TALENS， 和CRISPR/CAS9。需要可靠且经济实惠的冷冻保存方法来存档这些 模型，并确保其未来的供应。精子冷冻保存，这是一种简单而廉价的方法 进行，是保存转基因大鼠模型的理想选择；然而，成功的方法有限 使用冷冻大鼠精子的冷冻复苏使用的方法，如胞浆内单精子注射(ICSI)，这些方法是 超越了大多数研究实验室或转基因核心的手段，即使对有经验的人来说也是具有挑战性的 像RRRC这样的设施。进行体外受精(IVF)的能力，这是一种常见的、非常成功的 小鼠冷冻复苏的方法，将是大鼠冷冻复苏的理想方法。不幸的是，有限的 已发表的大鼠体外受精方案还没有可靠地被其他机构重复，包括RRRC。基于 初步数据显示，补充氨基酸对精子活力有有益影响，我们建议 测试添加不同氨基酸组合到培养液中，以测量这种补充的结果 精子运动、获能和体外受精。补充氨基酸的能力提高 冷冻/解冻精子用于解冻后精子活力和提高体外受精成功率 也要接受评估。这些研究将使用近交系和近交系的精子进行。 菌株。所产生的数据将指导优化大鼠精子冷冻保存和大鼠体外受精，从而提高 RRRC以经济高效的方式存档和复活大鼠模型的能力。

Abstract (Applied Research) The generation of novel genetically modified rat models has greatly accelerated with the availability of rat embryonic stem cells and the advent of nuclease-based genome-editing technologies such as ZFNs, TALENs, and CRISPR/Cas9. There is a need for reliable and cost-effective methods of cryopreservation to archive these models and ensure their future availability. Sperm cryopreservation, which is straightforward and inexpensive to perform, is ideal for preserving genetically modified rat models; yet, the limited methods for successful cryorecovery using frozen rat sperm employ methods, such as intracytoplasmic sperm injection (ICSI) that are beyond the means of most research laboratories or transgenic cores and are challenging even for experienced facilities like the RRRC. The ability to perform in vitro fertilization (IVF), which is a common and highly successful method of cryorecovery in the mouse, would be ideal for cryorecovery in the rat. Unfortunately, the limited published protocols for rat IVF have not been reliably repeatable by others, including the RRRC. Based on preliminary data suggesting amino acid supplementation has a beneficial impact on sperm motility, we propose to test the addition of various amino acid combinations to media to measure the outcome of this supplementation on sperm motility, capacitation and in vitro fertilization. The ability of amino acid supplementation to improve post-thaw sperm motility and increase in vitro fertilization success rates when frozen/thawed sperm is used will also be evaluated. These studies will be performed with spermatozoa from both outbred stocks and inbred strains. The data generated will guide optimization of rat sperm cryopreservation and rat IVF, thus enhancing the RRRC’s ability to archive and reanimate rat models in a cost-effective and efficient manner.