Common Human Stem Cell Lines for Inducible Gene Expression and Knockdown

描述（由申请人提供）：人类胚胎干细胞（hESC），像它们的小鼠对应物一样，提供了一种有用的工具来揭示正常和异常人类发育背后的细胞和分子事件。hESC的定向分化可以允许建立用于药物筛选和产生用于潜在再生医学的功能性细胞/组织的系统。与它们的小鼠对应物不同，hESC难以进行遗传修饰。对缓慢生长的hESC进行基因修饰的技术困难已经成为大多数实验室使用hESC进行其个人研究计划的重要障碍。我们建议工程师一个共同的人胚胎干细胞系与Cre重组介导的交换系统的条件基因表达和敲低。该提议是基于我们成功构建了携带可更换盒的组成型主hESC系。我们将首先通过相同的Cre重组介导的机制使用诱导型载体修饰组成型主hESC系（源自NIH Registry WA 09和WA 01），以构建具有用于转基因表达或敲低的可交换盒的主诱导型hESC系（Aim 1）。然后，我们将建立hESC系，以条件性表达神经原基因neurogenin 2（Ngn 2），并通过Cre介导的重组在独特的loxP位点敲低多能基因Oct 4，并证实靶基因表达可以在体外和移植到免疫缺陷小鼠后进行调控（Aim 2）。这些共同的hESC系将被存放在NIH赞助的国家干细胞库。这样一个有条件的主hESC线的可用性将提供一个灵活和容易的平台，在大多数实验室操作hESC的基础和应用研究。它最终将加速hESC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..