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• W883636071 abstract "We have developed a novel genetic platform enabling high-throughput identification of genetic targets in embryonic stem cells (ESC) of the mouse (Mamm Genome 20: 11, 2009). We propose to utilize this platform to identify genes causing cryotoxicity. The method consists of mass screening of mutated populations of ESC. Mutated cells are then exposed to a near-lethal concentration of a cryogenic agent or a stressful condition. Mutant ESC surviving exposure to the agent or stress grow and form colonies, which are picked and verified to be resistant to the stress. Hundreds of thousands of mutations can be screened simultaneously to identify those rare mutants causing resistance resulting from a mutational event. In our platform, mutations are generated by a transposable element called “PiggyBack” (PB). When activated by an appropriate transposase, the PB vectors move throughout the genome inserting themselves into the genome almost randomly and disrupting correct gene expression in a variety of ways. The sites of insertion and the genes disrupted by the PB element are identified by PCR off the ends of the PB element into the flanking DNA. The ability of the ESC to develop into mice (called pluripotency) is maintained by using a replica plating strategy so that a so-called “Master Plate” of the ESC are never exposed to a stressor. Thus, these mutant ESC can be used to generate intact, completely normal, adult mice, which can be used for organ harvest. These organs are composed entirely of genetically modified ESC, which can then be tested in organ transplants. For use in human (and perhaps in xenograft) transplants, drugs that block cytotoxicity will be developed. Genes and their protein products that lead to resistance become targets for developing drugs that should enhance organ viability during cryogenic storage. In preliminary results, it seems that we can establish conditions, using M22, that will completely kill the parental unmutated ESC. We have demonstrated that we can identify genes mediating resistance to cryotoxins and verify their role in this resistance. When we expose mutated ESC to the toxin, we find some cells forming colonies and these cells grow and divide under conditions that kill all unmutated cells. These ESC seem to maintain pluripotency. The sites of insertion are many and we have found many (some very unusual) targets for drug development. We hope to expand our studies to a variety of other stressful cryogenic agents and conditions. We also want to initiate drug screening and clinical chemistry, allowing us to move these methods to the organ transplantation community." @default.
• W883636071 created "2016-06-24" @default.
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• W883636071 date "2015-08-01" @default.
• W883636071 modified "2023-09-24" @default.
• W883636071 title "32. Eliminating toxicity during long-term cryogenic storage of human organs" @default.
• W883636071 doi "https://doi.org/10.1016/j.cryobiol.2015.05.038" @default.
• W883636071 hasPublicationYear "2015" @default.
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