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- W156832535 abstract "It has been shown that overexpression of four genes (Oct4, Sox2, c-Myc, Klf4) is sufficient to reprogram somatic cells into induced pluripotent stem (iPS) cells. To date, there are few viable reprogramming strategies that do not modify the genome of target cells, and even fewer that do not involve introduction of exogenous DNA. Herein, we describe a novel approach to specifically regulate expression of critical self-renewal and pluripotency genes in mouse embryonic stem cells, with the ultimate goal of generating iPS cells. We have constructed DRPs (designed regulatory proteins), which consist of a number of peptide domains. They are made of 6 zinc-finger (ZF) domains, as well as a nuclear localization signal (NLS), a cell- penetrating peptide (CPP), and a transeffect domain (TD). As proof of concept, we have conducted experiments using DRP-GFP fusion constructs to analyze DRP delivery and localization, and we have used the Oct4-DRP in order to test binding and functionality. We have established effective, inexpensive, high-yield protocols to express and purify DRPs targeting any gene. Through protein experiments, we have shown that the GFP-DRP fusion protein is translocated into the cell. In cDNA plasmid transfection experiments, we have shown that an altered version of this construct localizes in the nucleus. We have also demonstrated in vitro binding of two Oct4 DRPs to their respective probe sequences. Most importantly, we have demonstrated functionality of our Oct4 DRP under cellular conditions in HEK293 cells by demonstrating a 4- fold increase of expression in a reporter gene assay, when compared with controls. Though there are still difficulties to be solved, our work lays the foundation for a novel method to regulate genes that are important for generating iPS cells, as well as pluripotency and self -renewal in mouse embryonic stem cells. Because use of DRPs does not leave any lasting changes to the target cells' genome, our approach may prove valuable for future therapeutic use" @default.
- W156832535 created "2016-06-24" @default.
- W156832535 creator A5077904194 @default.
- W156832535 date "2009-01-01" @default.
- W156832535 modified "2023-09-27" @default.
- W156832535 title "Designing and characterizing artificial transcription factors targeting critical embryonic stem cell self-renewal and pluripotency genes in somatic cells" @default.
- W156832535 hasPublicationYear "2009" @default.
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