Common Human Stem Cell Lines for Inducible Gene Expression and Knockdown

描述(申请人提供)：人类胚胎干细胞(HESCs)，就像它们的小鼠一样，提供了一个有用的工具来揭示人类正常和异常发育的细胞和分子事件。HESCs的定向分化可以为潜在的再生医学建立药物筛选和生产功能细胞/组织的系统。与小鼠不同的是，hESCs很难从基因上进行修改。对生长缓慢的hESCs进行基因改造的技术困难已经成为大多数实验室使用hESCs进行个人研究计划的重大障碍。我们建议设计一个通用的hESC系，利用Cre-重组介导的交换系统进行有条件的基因表达和敲除。这一建议是基于我们成功地构建了一种携带可更换盒式磁带的构成主控hESC系列。我们将首先通过相同的Cre重组介导机制，通过使用可诱导载体对构成母体hESC系(来源于NIH注册表WA09和WA01)进行修饰，以构建具有可交换盒的母体可诱导的hESC系，用于转基因表达或敲除(目标1)。然后，我们将建立hESC系，有条件地表达神经源性基因Neurogenin 2(Ngn2)，并通过Cre介导的独特loxP位点的重组敲除多潜能基因Oct4，并证实目的基因的表达在体外和移植到免疫缺陷小鼠后都可以调节(目标2)。这些常见的hESC株将保存在NIH赞助的国家干细胞库中。这种有条件的hESC主生产线的提供将为大多数实验室的基础和应用研究提供一个灵活和容易的操作hESCs的平台。它最终将加速人类胚胎干细胞在再生医学中的潜在应用。

DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs), like their mouse counterparts, provide a useful tool to unveil cellular and molecular events underlying normal and abnormal human development. Directed differentiation of hESCs may allow establishing systems for pharmaceutical screening and producing functional cells/tissues for potential regenerative medicine. Unlike their mouse counterparts, hESCs are difficult to modify genetically. Technical difficulties in genetically modifying the slowly growing hESCs have become a significant barrier for most laboratories to proceed with their individual research programs using hESCs. We propose to engineer a common hESC line with the Cre-recombination mediated exchange system for conditional gene expression and knockdown. This proposal is based on our success in building a constitutive master hESC line that carries an exchangeable cassette. We will first modify the constitutive master hESC line (derived from NIH Registry WA09 and WA01) by using an inducible vector through the same Cre recombination mediated mechanism to build master inducible hESC lines with exchangeable cassette for transgene expression or knockdown (Aim 1). We will then build hESC lines to conditionally express a neurogenic gene neurogenin 2 (Ngn2) and knock down a pluripotent gene Oct4 through Cre mediated recombination at the unique loxP sites, and confirm that the target gene expression can be regulated in vitro and following transplantation into the immune deficient mice (Aim 2). These common hESC lines will be deposited in the NIH-sponsored National Stem Cell Bank. Availability of such a conditional master hESC line will provide a flexible and ease platform to manipulate hESCs in most laboratories for both basic and applied researches. It will ultimately speed up the potential use of hESCs in regenerative medicine..