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Transcript
KEVIN HIOM Galway 2010 Chapter 1: Epistasis for beginners Basic principles of DT40 DT40: A genetically tractable eukaryotic cell line DT40 • Genetically tractable • Good model for genome stability in mammals • Complementation by human genes • Good database versus humans Genetically tractable DT40 Phenotypic analysis Knocking out or mutating genes and looking at cellular function Mapping genetic pathways Combining mutations- epistasis Structure/function analysis/ cell biology Complementation, proteomics Genetic regulation Reporter assays All these require manipulation of the genome Integrate DNA Target DNA * Alter DNA * Remove DNA Genetic Recombination is our tool Random Integration- non homologous end joining Targeted Integration- Homologous/Homeologous recombination Site specific recombination Non Homologous End Joining-Random integration Ku, DNA-PKcs, LigIV, Advantages Simple Relatively high frequency Disadvantages Potential uncharacterised genetic effect Multiple integration Shut down of expression Homologous recombination- site specific integration, gene disruption, mutation DNA End Resection Mre11/RAD50/NBS1, CtIP, Exo1 Strand Invasion RAD51 Holliday Junctions Branch Migration RAD51BCD Resolution Slx1/4, GEN1 Homologous recombination Homologous Recombination Advantage s Acurate/error free Introduction of multiple changes Disdvantages Easy to introduce errors Aberrant recombination Neighbouring sequences Epistasis difficult for HR genes Site specific recombination- cre/lox ATAACTTCGTATAGCATACATTATACGAAGTTAT LOXP Site specific recombination- re-using antibiotic resistance drugr Cre recombinase synapsis excision Site specific recombination Courtesy of the National Library of Medicine (NLM) Understanding recombination is the key to manipulating the DT40 genome Words of warning 3 copies of chromosome 2 Genomes are ‘plastic’- Don’t culture for too long
