In vitro induction,expression and selection of thioguanine-resistant mutants with human T-lymphocytes |
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| Institution: | 1. Department of Design and Environmental Analysis, Cornell University, Ithaca, NY, United States;2. Human Development, Cornell University, Ithaca, NY, United States;3. Institute of Architectural Sciences, Vienna University of Technology, Vienna, Austria;4. Parkin Architects, Toronto, Canada;1. School of Electric Power Engineering, China University of Mining and Technology, Xuzhou 221116, PR China;2. Key Laboratory of Energy Thermal Conversion and Control of Ministry of Education, School of Energy and Environment, Southeast University, Nanjing 210096, PR China;3. Department of Environmental Science and Technology, Tokyo Institute of Technology, Kanagawa 226-8503, Japan;1. School of Water Resources and Hydropower Engineering, North China Electric Power University, Beijing 102206, China;2. China Inst Water Resources & Hydropower Res, State Key Lab Simulat & Regulat Water Cycle River, Beijing 100038, China;1. Pharmacoinformatics Department, National Institute of Pharmaceutical Education and Research (NIPER), Hajipur 844102, India;2. BioMedical Informatics Division, Rajendra Memorial Research Institute of Medical Sciences, Agam Kuan, Patna 800007, India;3. Biochemistry Department, Rajendra Memorial Research Institute of Medical Sciences, Agam Kuan, Patna 800007, India |
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| Abstract: | Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G0 phase peripheral blood mononuclear cells from a single individual. After explosure to γ-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TGr) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 × 104 cells/well. The development of this in vitro assay should allow direct study of susceptibility to γ-irradiation in the human population in terms of both cytotoxicity and mutation induction. |
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