Sperm Stem Cell Libraries for Biological Research

描述（由申请人提供）：遗传修饰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。鉴于这种潜力，我们以前建立的协议，用于分离，繁殖，遗传修饰和确定大鼠精原细胞培养作为一种新的应用类型的生殖系干细胞的生殖系传输率。我们最初选择大鼠作为这些研究的物种，是因为其作为人类疾病的实验室规模哺乳动物模型的流行性。这是基于需要新的协议来突变大鼠的基因，以研究使用小鼠无法鲁棒建模的生物过程。在初步研究中，睡美人基因陷阱转座子被成功地用于破坏精原细胞系中的转录序列。因此，本项目的具体目标将开发一种新的和强大的“不育睾丸互补模型”，其中供体精原细胞和不育受体作为载体，最有效地通过大鼠生殖系传递供体转基因。该系统最大限度地直接从大鼠精原细胞的突变基因的种系传输，因此，旁路的中间需要产生和繁殖嵌合体/chlmairic后代。在特定目标1中，将使用转座子（即b-Geo、Venus、dtTomato）将不同常用选择标记整合到大鼠精原基因组中后，定义其生殖系传递率。在特定目标2中，将使用两种不同的转座子系统（即Sleeping Beauty和PiggyBac）在大鼠精原细胞培养物以及用精原细胞培养物生产的活大鼠中确定转录基因的突变谱，所述转座子系统被设计用于产生条件性和可逆性敲除大鼠。在具体目标3中，将使用全基因组测序方法对大鼠精原细胞文库中的大量基因敲除进行充分注释。将筛选文库中所选的靶向生殖系，然后将其用于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内预先定义条件基因陷阱突变将使最用户友好和最具成本效益的策略可用于靶向大鼠中的破坏基因。 公共卫生相关性（由申请人提供）：该项目将使用当代技术为科学界建立前所未有的大鼠遗传模型资源。该资源将极大地扩展生物医学的潜力和发现，基本上在生命科学的每个领域，通过允许研究人员使用大鼠作为遗传模型研究基因功能和人类健康之间的关系。

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.