A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.

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 muta...

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Autores principales:	Stephen J Pettitt, Farah L Rehman, Ilirjana Bajrami, Rachel Brough, Fredrik Wallberg, Iwanka Kozarewa, Kerry Fenwick, Ioannis Assiotis, Lina Chen, James Campbell, Christopher J Lord, Alan Ashworth
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e0b684f60ba24e7a892ea6f6dcb1a2c1
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spelling	oai:doaj.org-article:e0b684f60ba24e7a892ea6f6dcb1a2c12021-11-18T07:47:54ZA genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.1932-620310.1371/journal.pone.0061520https://doaj.org/article/e0b684f60ba24e7a892ea6f6dcb1a2c12013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23634208/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Genetic 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.Stephen J PettittFarah L RehmanIlirjana BajramiRachel BroughFredrik WallbergIwanka KozarewaKerry FenwickIoannis AssiotisLina ChenJames CampbellChristopher J LordAlan AshworthPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 4, p e61520 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Stephen J Pettitt Farah L Rehman Ilirjana Bajrami Rachel Brough Fredrik Wallberg Iwanka Kozarewa Kerry Fenwick Ioannis Assiotis Lina Chen James Campbell Christopher J Lord Alan Ashworth A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.
description	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.
format	article
author	Stephen J Pettitt Farah L Rehman Ilirjana Bajrami Rachel Brough Fredrik Wallberg Iwanka Kozarewa Kerry Fenwick Ioannis Assiotis Lina Chen James Campbell Christopher J Lord Alan Ashworth
author_facet	Stephen J Pettitt Farah L Rehman Ilirjana Bajrami Rachel Brough Fredrik Wallberg Iwanka Kozarewa Kerry Fenwick Ioannis Assiotis Lina Chen James Campbell Christopher J Lord Alan Ashworth
author_sort	Stephen J Pettitt
title	A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.
title_short	A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.
title_full	A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.
title_fullStr	A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.
title_full_unstemmed	A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.
title_sort	genetic screen using the piggybac transposon in haploid cells identifies parp1 as a mediator of olaparib toxicity.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/e0b684f60ba24e7a892ea6f6dcb1a2c1
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A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.

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