IMPROVED LENTIVIRAL VECTORS FOR PRIMATE ES CELLS

描述（由申请人提供）：ES细胞可以无限制地增殖，即使在长时间培养后，也保留形成从心肌到神经到血液的细胞的能力;可能是构成身体的任何细胞类型。 非人灵长类动物（NHP），即恒河猴和普通绒猴，以及人类ES细胞的最新衍生对人类发育生物学，药物发现，药物测试和移植医学具有广泛的意义。 由于人类和旧世界猴之间的胚胎学相似性，恒河猴ES细胞和恒河猴为开发基于人类ES细胞的疗法提供了极其准确、必要的模型系统。 灵长类ES细胞的有效遗传操作对于以下方面是必不可少的：1）阐明分化过程中和特定分化细胞中的基因功能; 2）通过操纵转录因子指导ES细胞分化为特定谱系; 3）通过引入选择性标记从ES细胞衍生物的混合群体中纯化所需的分化细胞类型; 4）使用灵长类动物ES细胞的分化衍生物作为基因治疗的载体;和5）调节对移植的ES细胞衍生物的免疫应答。 不幸的是，灵长类动物的胚胎干细胞是非常难以复制的。 常规用于小鼠ES细胞的转染方法不能用于灵长类ES细胞。 最近，测试了一系列具有内部启动子的假型化的自失活慢病毒载体。 这些是第一个测试的载体，允许恒河猴和绒猴ES细胞系稳定表达外源基因的衍生。 为了改进这些用于灵长类ES细胞的载体，并将它们用作剖析灵长类ES细胞自我更新机制的工具，将实现以下具体目的：1）通过测试绝缘子序列和支架附着区的作用，产生在灵长类ES细胞及其分化的衍生物中具有改进的长期、位点非依赖性表达的慢病毒载体; 2）使用功能性ES细胞筛选来分离促进慢病毒载体的长期、整合位点非依赖性表达的新基因组DNA片段; 3）使用慢病毒载体测试参与其他干细胞自我更新的特定基因的作用（β-连环蛋白、ID-1、N-myc、Notch、STAT-3）在促进灵长类ES细胞自我更新中的作用;和4）使用慢病毒cDNA表达克隆策略来鉴定由成纤维细胞或由ES细胞自身产生的促进ES细胞自我更新的新基因。

DESCRIPTION (provided by applicant): ES cells can proliferate without limit, and even after prolonged culture, retain the ability to form cells ranging from cardiac muscle to nerve to blood; potentially any cell type that makes up the body. The recent derivation of nonhuman primate (NHP), i.e. rhesus monkey and common marmoset, and human ES cells has widespread implications for human developmental biology, drug discovery, drug testing, and transplantation medicine. Because of the close embryological similarities between humans and old world monkeys, rhesus monkey ES cells and rhesus monkeys provide an extremely accurate, necessary model system for developing human ES cell-based therapies. The efficient genetic manipulation of primate ES cells is essential to: 1) elucidate gene function both during differentiation and in specific differentiated cells; 2) direct the differentiation of ES cells to specific lineages by the manipulation of transcription factors; 3) purify desired differentiated cell types from a mixed population of ES cell derivatives by introducing selectable markers; 4) use the differentiated derivatives of primate ES cells as vehicles for gene therapy; and 5) modulate the immune response to transplanted ES cell derivatives. Unfortunately, primate ES cells are extremely difficult to transfect. Transfection methods routinely used for mouse ES cells fail for primate ES cells. Recently, a series of pseudotyped, self-inactivating lentiviral vectors with internal promoters were tested. These are the first vectors tested that allow the derivation of rhesus and marmoset ES cell lines stably expressing a foreign gene. To improve these vectors for primate ES cells, and to use them as tools to dissect mechanisms of primate ES cell self-renewal, the following Specific Aims will be accomplished to: 1) generate lentiviral vectors with improved long term, site independent expression in primate ES cells and their differentiated derivatives by testing the effects of insulator sequences and scaffold attachment regions; 2) use a functional ES cell screen to isolate novel genomic DNA fragments that promote long term, integration site independent expression of lentiviral vectors; 3) use lentiviral vectors to test the role of specific genes involved in the self-renewal of other stem cells (Beta-catenin, ID-1, N-myc, Notch, STAT-3) in promoting the self-renewal of primate ES cells; and 4) use a lentiviral cDNA expression cloning strategy to identify novel genes produced by fibroblasts, or by the ES cells themselves that promote ES cell self-renewal.