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.. ? ? ?
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