Sperm Stem Cell Libraries for Biological Research

用于生物学研究的精子干细胞文库

基本信息

批准号：
8687761
负责人：
F. Kent Hamra
金额：
$ 56.13万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-07 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8687761
关键词：
Biological Process Biological Sciences Breeding Communities Complex Computer Simulation Data Effectiveness Frequencies Gene Expression Gene Targeting Genes Genetic Genetic Models Genome Genomic Library Genotype Grant Health Human In Vitro Individual Knock-out Laboratories Libraries Life Link Maps Massive Parallel Sequencing Measures Modeling Modification Monoclonal Antibody R24 Mus Mutagenesis Mutate Mutation Pilot Projects Principal Investigator Protocols documentation Rattus Reporting Research Personnel Resources Sleep Sleeping Beauty Spermatogonia Stem cells Sterility Structure System Techniques Technology Testis Transgenes Transplantation Venus base biological research cost effective cost effectiveness design gene function genome sequencing genome-wide human disease in vivo innovation interest knockout gene mutant novel sperm cell transmission process user-friendly vector

项目摘要

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.

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，我们之前建立了用于分离、繁殖、基因修饰和确定大鼠精原细胞培养物的种系传播率的方案，作为一种新应用的种系干细胞类型。我们最初选择大鼠作为这些研究的物种，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。这是基于需要新的方案来突变大鼠的基因，以研究无法使用小鼠进行稳健建模的生物过程。在试点研究中，睡美人基因捕获转座子被成功地用于破坏精原细胞系中的转录序列。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，通过大鼠种系最有效地传递供体转基因。该系统最大化了直接来自大鼠精原细胞的突变基因的种系传递，因此绕过了产生和繁殖嵌合/chmairic后代的中间需要。在具体目标 1 中，将针对不同的常用选择标记在使用转座子（即 b-Geo、Venus、dtTomato）整合到大鼠精原基因组中后定义种系传播率。在具体目标 2 中，将在大鼠精原细胞培养物以及用精原细胞培养物产生的活体大鼠中确定转录基因的突变谱，使用两种不同的转座子系统（即睡美人和 PiggyBac），旨在产生条件性和可逆性基因敲除大鼠。在具体目标 3 中，将使用全基因组测序方法对大鼠精原细胞文库中的大量基因敲除进行完整注释。该文库将筛选选定的目标种系，然后用于生成所需的突变大鼠。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陷阱突变将使最用户友好且最具成本效益的策略可用于靶向大鼠中的破坏基因。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Spermatogonial Gene Networks Selectively Couple to Glutathione and Pentose Phosphate Metabolism but Not Cysteine Biosynthesis.

DOI：
10.1016/j.isci.2020.101880
发表时间：
2021-01-22
期刊：
iScience
影响因子：
5.8
作者：
Prokai D;Pudasaini A;Kanchwala M;Moehlman AT;Waits AE;Chapman KM;Chaudhary J;Acevedo J;Keller P;Chao X;Carr BR;Hamra FK
通讯作者：
Hamra FK

NRG1 and KITL Signal Downstream of Retinoic Acid in the Germline to Support Soma-Free Syncytial Growth of Differentiating Spermatogonia.