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Development of the high throughput mammalian PIG-A gene mutation assay in vitro. / Benjamin James Rees
Swansea University Author: Benjamin James Rees
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The field of genetic toxicology has recently undergone reform which has limited or banned the use of animal models within a number of different industries (cosmetics). Consequently, greater emphasis has been placed on developing novel, highly sensitive, in vitro test systems which can generate robus...
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The field of genetic toxicology has recently undergone reform which has limited or banned the use of animal models within a number of different industries (cosmetics). Consequently, greater emphasis has been placed on developing novel, highly sensitive, in vitro test systems which can generate robust data to aid regulatory hazard and risk assessment.The main aims of this project were i) to develop a highly sensitive and specific, high throughput mammalian in vitro PIG-A gene mutation assay to enable quantitative dose response modelling and further investigate the potential use of in vitro data within human health assessment, ii) Investigate the genotype to phenotype relationship, a potentially delaying step within future OECD guideline drafting for the current in-vivo Pig-a mutation assay and iii) help develop and optimise a preliminary comprehensive human PIG-A biomonitoring platform.During in-vitro and ex-vivo PIG-A assay development, flow cytometry was the fundamental technique utilised. Multiple additional laser excitation platforms were evaluated for use, including Amnis ImageStream ™ and laser scanning confocal. Proteomic as well as genomic techniques were used during the supplementary investigations surrounding assay development, with microbiological groundings throughout.The finalised in-vitro assay protocol was established within human, metabolically active, MCL-5 cells. Using the refined assay design, proof of principle experimentations were able to show the potential for future quantitative work and the general promise with this novel approach. The genotype to phenotype relationship validation is currently still on-going following the preliminary work described herein and recent publications. The ex-vivo human PIG-A assay platforms were shown to require further optimisation in terms of sensitivity, excluding red blood cells, but showed good aptitude for future use.Currently it looks promising that further refinement could lead to a comprehensive high content, high-throughput assay system with the potential to be used within future hazard and risk assessment
Faculty of Medicine, Health and Life Sciences