Sperm Stem Cell Libraries for Biological Research

描述(申请人提供)：从基因上修改或破坏大鼠基因表达的经济方法将广泛适用于加速科学发现。鉴于这一潜力，我们以前建立了分离、繁殖、遗传修改和测定大鼠精原培养的生殖系传播率的方案，作为一种新的应用类型的生殖系干细胞。我们最初选择大鼠作为这些研究的物种，是因为它作为实验室规模的人类疾病哺乳动物模型很受欢迎。这是基于需要新的方案来突变大鼠的基因，以研究不能像用小鼠那样健壮地建模的生物过程。在初步研究中，睡美人基因陷阱转座子被成功地用于扰乱精原细胞系的转录序列。因此，当前项目的具体目标将开发一种新的、强大的“不育-睾丸-互补模型”，在该模型中，捐赠者精原细胞和不育接受者作为载体，通过大鼠生殖系最有效地传播捐赠者的转基因。该系统使直接来自大鼠精原细胞的突变基因的种系传递最大化，因此，绕过了产生和繁殖马赛克/黄蜂后代的中间需要。在具体目标1中，将利用转座子(即b-geo、Venus、dtTomato)将不同常用的可选标记整合到大鼠精原基因组中后，定义它们的生殖系传播率。在具体目标2中，将使用两种不同的转座子系统(即睡美人和猪Bac)，在大鼠精原细胞培养物和用精原细胞培养物产生的活体大鼠中测定转录基因的突变谱，以产生条件和可逆的基因敲除大鼠。在具体目标3中，将使用全基因组测序方法对大鼠精原文库中的大量基因敲除进行完全注释。文库将被筛选出选定的目标生殖系，然后将被用来产生所需的突变大鼠。因此，在突变精原细胞的复杂基因组文库中预先定义条件性基因陷阱突变将使用户友好和最具成本效益的策略能够针对大鼠的干扰基因。 公共卫生相关性(由申请者提供)：该项目将使用当代技术为科学界建立前所未有的老鼠遗传模型资源。通过允许研究人员使用大鼠作为遗传模型来研究基因功能与人类健康之间的关系，将极大地扩展生命科学几乎每个领域的生物医学潜力和发现。

DESCRIPTION (provided by applicant): Economical approaches to genetically modify or disrupt the expression of genes in rats would be widely applicable to accelerating scientific discovery. In view of this potential, we previously established protocols for isolating, propagating, genetically modifying and determining the germline transmission rates of rat spermatogonial cultures as a newly applied type of germline stem cell. We initially chose the rat as a species for these studies due to its popularity as a laboratory scale mammalian model for human disease. This is based on the need for new protocols to mutate genes in rats to study biological processes that are not as robustly modeled using mice. In pilot studies, Sleeping Beauty gene-trap transposons were successfully used to disrupt transcribed sequences in spermatogonial lines. Accordingly, Specific Aims of the current project will exploit a new and robust "sterile-testis-complementation model" in which donor spermatogonia and sterile recipients serve as vectors to most effectively transmit donor transgenes through the rat germline. This system maximizes germline transmission of mutatant genes directly from rat spermatogonia, and therefore, by-passes the intermediate need to produce and breed mosaic/chlmairic progeny. In Specific Aim 1, germline transmission rates will be defined for different commonly applied selectable markers after their integration into rat spermatogonial genomes using transposons (i.e. b-Geo, Venus, dtTomato). In Specific Aim 2, mutational spectra of transcribed genes will be determined in both, cultures of rat spermatogonia, plus live rats produced with the spermatogonial cultures, using two different transposon systems (i.e. Sleeping Beauty & PiggyBac) designed to generate conditional and reversible knockout rats. In specific Aim 3, large numbers of gene knockouts within rat spermatogonial libraries will be fully annotated using whole genome sequencing approaches. The library will be screened for the chosen targeted germlines, which will then be used to generate the desired mutant rats. Thus, pre-defining conditional gene-trap mutations within complex genomic libraries of mutant spermatogonia will enable the most user friendly and cost-effective strategy available to target the disruption genes in the rat. PUBLIC HEALTH RELEVANCE (provided by applicant): This project will use contemporary technologies to build an unprecedented resource of rat genetic models for the scientific community. The resource of will dramatically expand the biomedical potential and discovery in essentially every field of the life sciences by allowing researchers to study relationships between gene function and human health using the rat as a genetic model.