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