Project

应用研究（Applied Research） 新型转基因大鼠模型的产生大大加快了大鼠的可用性， 胚胎干细胞和基于核酸酶的基因组编辑技术的出现，如ZFN，TALEN， CRISPR/Cas9需要可靠且具有成本效益的冷冻保存方法来保存这些 模型，并确保其未来的可用性。精子冷冻保存，这是直接和廉价的 执行，是理想的保存转基因大鼠模型;然而，有限的方法，成功 使用冷冻的大鼠精子的冷冻复苏采用诸如胞浆内精子注射（ICSI）的方法， 这超出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一个挑战。 像RRRC这样的设施。进行体外受精（IVF）的能力，这是一种常见且非常成功的方法。 小鼠冷冻复苏的方法，将是理想的大鼠冷冻复苏。不幸的是， 已发表的大鼠体外受精方案还没有被其他人可靠地重复，包括RRRC。基于 初步数据表明，补充氨基酸对精子活力有有益的影响，我们建议 测试向培养基中添加各种氨基酸组合以测量这种补充的结果 精子活力获能和体外受精补充氨基酸的能力提高 解冻后的精子活力和增加体外受精成功率时，使用冷冻/解冻精子将 也被评价。这些研究将使用来自远交原种和近交原种的精子进行 菌株所产生的数据将指导大鼠精子冷冻保存和大鼠IVF的优化，从而提高 RRRC以具有成本效益和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.