Genetic perturbation screens have the
potential to dissect a wide range of cellular phenotypes. Such screens have
historically been difficult in diploid mammalian cells. The recent derivation
of haploid embryonic stem cells provides an opportunity to cause loss of
function mutants with a random mutagen in a mammalian cell with a normal
genetic background. We describe an approach to genetic screens that exploits
the highly active piggyBac transposon in haploid mammalian cells. As an example
of haploid transposon (HTP) screening, we apply this approach to identifying
determinants of cancer drug toxicity and resistance. In a screen for
6-thioguanine resistance we recovered components of the DNA mismatch repair
pathway, a known requirement for toxicity. In a further screen for resistance
to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we
recovered multiple Parp1 mutants. Our results show that olaparib toxicity to
normal cells is mediated predominantly via Parp1, and suggest that the clinical
side effects of olaparib may be on target. The transposon mutant libraries are
stable and can be readily reused to screen other drugs. The screening protocol
described has several advantages over other methods such as RNA interference:
it is rapid and low cost, and mutations can be easily reverted to establish
causality.
Source: A Genetic Screen Using the
PiggyBac Transposon in Haploid Cells Identifies Parp1 as a Mediator of Olaparib
Toxicity. Pettitt SJ, Rehman FL, Bajrami I, Brough R, Wallberg F, Kozarewa I,
Fenwick K, Assiotis I, Chen L, Campbell J, Lord CJ, Ashworth A (Alan.Ashworth@icr.ac.uk). PLoS One.
2013 Apr 25;8(4):e61520.
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