Naturally sterile hybrid mice for the production of embryo transfer recipients

转基因（GM）小鼠模型提供了一个强大的手段归因于DNA序列的功能。随着DNA变异和突变如何导致疾病的研究进展，对转基因小鼠模型的需求增加。最近，新的CRISPR技术帮助了这一需求，该技术允许以前所未有的效率生产模型。为了生成和分配转基因小鼠模型，胚胎在体外进行操作和/或培养，并需要转移到受体雌性小鼠体内，然后由受体雌性小鼠作为转移胚胎的养母。这些寄养雌性小鼠是通过与不育雄性小鼠交配来制备的，这使它们处于一种称为假孕的状态，使它们能够接受胚胎并将其带到足月。传统上，用于此目的的雄性小鼠通过输精管切除术而不育，这需要将雄性小鼠麻醉并进行手术。恢复后，在用于产生用于胚胎移植的受体雌性小鼠之前，测试雄性小鼠的不育性。作为替代方案，已知某些基因修饰的小鼠品系，其中基因敲除或转基因导致雄性不育，并且这些小鼠品系可用于代替输精管切除的雄性。显然，这些遗传不育的雄性小鼠提供了一个很好的替代方案，避免了进行手术输精管切除术的需要，然而，这些遗传不育小鼠品系的繁殖需要大量的繁殖程序，并且许多小鼠在其生产中被浪费。此外，用于实现遗传不育的一些遗传构建体不是100%可靠的，并且这些构建体的随机失活有时导致恢复的育性。同样，小鼠在使用前必须进行无菌性测试。我们一直在探索杂交小鼠，这是当两个不同品系或物种的小鼠交配在一起时产生的后代。某些杂交种表现出天然的雄性不育，存在于100%的雄性中。这些现象是从自然界中很好地认识到的-一个常见的例子是当马和驴杂交时产生的不育骡子。在初步实验中，我们使用这些自然不育的杂交种来生产胚胎移植受体，发现它们和标准的输精管切除雄性一样善于生产寄养母亲。使用这些不育的杂种代表了一个重要的3R影响，因为，类似于遗传不育的男性，手术输精管切除术不再需要。然而，当与遗传不育雄性相比时，天然不育杂交种的产生要有效得多，因为由杂交种杂交产生的所有雄性都是不育的并且可以使用。相反，遗传不育的男性是无效的生产，有相当大的waste.We计划继续我们的工作与不育杂交为此目的，但想传播的方法，以其他研究中心，并已要求合作者在曼彻斯特大学和玛丽里昂中心，哈威尔，测试这些不育杂交对标准输精管切除男性和遗传不育男性的性能。通过在三个独立的地点确认这种方法，我们希望说服世界各地生产和分发转基因小鼠的中心采用这种策略，并希望冷冻保存许多杂交胚胎，以促进这项技术的转移。此外，我们计划确定雌性杂交小鼠是否可以作为胚胎移植受体，这意味着生产不育杂交小鼠不会浪费小鼠。

Genetically modified (GM) mouse models provide a powerful means of attributing function to DNA sequences. As investigations of how DNA variation and mutation contributes to disease advance, demand for GM mouse models increases. Recently, this demand has been aided by the new CRISPR technology, which permits the production of models at unprecedented efficiencies.To generate and distribute GM mouse models, embryos are manipulated and/or cultured in vitro and require transfer into recipient female mice who then act as foster mothers for the transferred embryos. These foster females are prepared by breeding with sterile male mice, which renders them in a state known as pseudopregnancy, enabling them to receive embryos and carry them to term. Traditionally the male mice used for this purpose have been made sterile by vasectomy, which requires that the male mice be anaesthetized and surgery performed. After recovery, the male mice are tested for sterility prior to being used to generate recipient female mice for embryo transfer. As an alternative, certain genetically modified mice strains are known where the gene knock-out or transgene results in male sterility and these can be used instead of vasectomized males. Clearly, these genetically sterile male mice provide a good alternative, avoiding the need for performing surgical vasectomy, however the breeding of these genetically sterile mice lines requires lots of breeding procedures and many mice are wasted in their production. Furthermore, some of the genetic constructs used to achieve genetic sterility are not 100% reliable and random inactivation of these constructs sometimes results in a restored fertility. Again, mice must then be tested for sterility prior to use. We have been exploring hybrid mice, which are the offspring that are produced when two mice of different strains or species are mated together. Certain hybrids show a natural male sterility that is present in 100% of the males. These phenomenon is well recognized from nature - a common example would be the sterile mule that is generated when a horse and a donkey interbreed. In preliminary experiments, we have used these naturally sterile hybrids for the production of embryo transfer recipients and find that they are just as good at producing foster mothers as the standard vasectomized males. The use of these sterile hybrids represents an important 3Rs impact, as, similar to genetically sterile males, surgical vasectomies are no longer needed. However, when compared to genetically sterile male, the production of naturally sterile hybrids is much more efficient as all males resulting from the hybrid interbreeding are sterile and can be used. In contrast genetically sterile males are inefficiently produced and there is considerable wastage.We plan to continue our work with sterile hybrids for this purpose, but would like to disseminate the method to other research centres and have asked collaborators at the University of Manchester and at the Mary Lyons Centre, Harwell, to test the performance of these sterile hybrids against standard vasectomized males and genetically sterile males. With a confirmation of this approach at three independent sites, we then hope to convince the centres world-wide who produce and distribute GM mice to adopt this strategy and hope to cryopreserve many hybrid embryos to facilitate the transfer of this technology. In addition, we plan to establish whether the female hybrids could serve as embryo transfer recipients, meaning that there would be no wastage of mice from the production of the sterile hybrids.