This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To generate lentiviral particles with improved long term site dependent expression in primate ES cells and their derivatives and to test specific genetic markers for their role in self renewal. In order to efficiently deliver exogenous genes into human embryonic stem cells (hESCs), we've optimized a lentiviral expression system: 1) we decreased the vector size to improve the cloning of various genes;2) we developed different expression vectors with different promoters such as EF1a promoter and PGK promoter to allow different levels of transgene expression, along with different drug-selection markers such as neomycin phosphotransferase and puromycin-N-acetyl-transferase;3) We optimized lentiviral packaging protocols, allowing us to obtain viral particles with consistent high titers. We have successfully produced 14 new lentiviral constructs using genes found to be essential for pluripotency in ES cells. We have also produced lentiviral particles from these 14 new constructs. Using these lentiviral particles, we were able to successfully reprogram differentiated skin cells back to ES-like cells. The new cells are called ips cells. This research used WNPRC Stem Cell Resources, ITSS services and federally approved human ES cell lines.
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