Sperm Stem Cell Libraries for Biological Research

描述（由申请人提供）：对大鼠进行基因修饰或破坏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，我们先前建立了分离、繁殖、基因修饰和确定大鼠精原培养物作为一种新应用的种系干细胞的种系传输率的方案。我们最初选择大鼠作为这些研究的物种，是因为它作为人类疾病的实验室规模哺乳动物模型很受欢迎。这是基于对大鼠基因突变的新方案的需求，以研究生物过程，而这些过程在小鼠身上没有得到强有力的模拟。在初步研究中，睡美人基因诱捕转座子被成功地用于破坏精原系的转录序列。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”，其中供体精原细胞和不育受体作为载体，最有效地通过大鼠种系传播供体转基因。该系统最大限度地提高了突变基因直接从大鼠精原细胞的种系传播，因此，绕过了产生和繁殖嵌合体/衣原体后代的中间需要。在Specific Aim 1中，将定义不同的常用选择标记在使用转座子（即b-Geo， Venus, dtomato）整合到大鼠精原基因组后的种系传播率。在Specific Aim 2中，将使用两种不同的转座子系统（即睡美人和PiggyBac）来产生条件和可逆敲除大鼠，在大鼠精原细胞培养物和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。在特定的Aim 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充分注释。该文库将筛选选定的目标生殖系，然后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中预先定义条件基因诱捕突变，将使针对大鼠中破坏基因的最用户友好和最经济有效的策略成为可能。

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.