Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells

Pettitt, Stephen J.; Krastev, Dragomir B.; Pemberton, Helen; Fontebasso, Yari; Frankum, Jessica; Rehman, Farah L.; Brough, Rachel; Song, Feifei; Bajrami, Ilirjana; Rafiq, Rumana; Wållberg, Fredrik; Kozarewa, Iwanka; Fenwick, Kerry; Armisen-Garrido, Javier; Swain, Amanda; Gulati, Aditi; Campbell, James; Ashworth, Alan; Lord, Christopher J.

doi:10.1038/sdata.2017.20

Cited by 15 publications

(16 citation statements)

References 31 publications

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“…Thus, haploid ESCs enable the isolation of knockout phenotypes in a single round of mutagenesis in a scalable manner. In another application, mutagenized haploid ESCs were screened for drug sensitivity phenotypes [81]. In this study ~100,000 mutant cell clones were generated by a gene trap PB transposon carrying an SA-IRES-neo cassette and a random barcode sequence, whose recovery by next generation sequencing enables quantitation of the number of cells bearing each barcode, and hence each transposon insertion.…”

Section: Transposons and Functional Genomicsmentioning

confidence: 99%

“…In this study ~100,000 mutant cell clones were generated by a gene trap PB transposon carrying an SA-IRES-neo cassette and a random barcode sequence, whose recovery by next generation sequencing enables quantitation of the number of cells bearing each barcode, and hence each transposon insertion. These mutant cell pools were then exposed to 13 different drugs followed by an assessment of barcode abundance in order to identify mutations that conferred sensitivity to inhibitors of topoisomerases, PARP and HSP90 [81]. The feasibility of introducing large numbers of mutagenic transposon insertions has also been demonstrated in haploid ESCs established from rats by gene trapping with PB vectors carrying an antibiotic selection marker [82].…”

Section: Transposons and Functional Genomicsmentioning

confidence: 99%

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Transposons As Tools for Functional Genomics in Vertebrate Models

2017

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Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles and providing insight into genetic networks and pathways based on phenotypes. For example, the Sleeping Beauty transposon has become highly instrumental to induce tumors in experimental animals in a tissue-specific manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice and rats.

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Section: Transposons and Functional Genomicsmentioning

confidence: 99%

Section: Transposons and Functional Genomicsmentioning

confidence: 99%

Transposons As Tools for Functional Genomics in Vertebrate Models

2017

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“…Given the cell identity and genomic instability of KBM7 cells, haploid ES cells should have a broader application in genetic screening, such as the one for ‘exit-from-pluripotency’ factors shown here, which obviously cannot be conducted on KBM7 cells. Moreover, targeted mutations or reporter constructs can easily be introduced into haploid ES cells ( 19 , 24 , 26 , 27 ), which should be beneficial for designing more precise genetic screens. For example, via the knock-in of a well-defined lineage-commitment reporter into haploid ES cells before the construction of arrayed mutant libraries, specific differentiation regulators for a particular cell type could be identified.…”

Section: Discussionmentioning

confidence: 99%

“…However, all these screens were performed in mixed pools of a vast number of mutant cells, indicating that the null mutants of interest must be selected either positively using a lethal drug or indirectly using an artificial cellular reporter construct. Recently, a genome-wide barcode transposon screen was used to identify drug-sensitive phenotypes, but the screen was still performed in mixed pools ( 24 ). Thus, the application of haploid ES cells in genetic screening remains limited.…”

Section: Introductionmentioning

confidence: 99%

Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens

Guang

Wang

Liu

et al. 2017

Nucleic Acids Research

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Forward genetic screens using mammalian embryonic stem (ES) cells have identified genes required for numerous cellular processes. However, loss-of-function screens are more difficult to conduct in diploid cells because, in most cases, both alleles of a gene must be mutated to exhibit a phenotype. Recently, mammalian haploid ES cell lines were successfully established and applied to several recessive genetic screens. However, all these screens were performed in mixed pools of mutant cells and were mainly based on positive selection. In general, negative screening is not easy to apply to these mixed pools, although quantitative deep sequencing of mutagen insertions can help to identify some ‘missing’ mutants. Moreover, the interplay between different mutant cells in the mixed pools would interfere with the readout of the screens. Here, we developed a method for rapidly generating arrayed haploid mutant libraries in which the proportion of homozygous mutant clones can reach 85%. After screening thousands of individual mutant clones, we identified a number of novel factors required for the onset of differentiation in ES cells. A negative screen was also conducted to discover mutations conferring cells with increased sensitivity to DNA double-strand breaks induced by the drug doxorubicin. Both of these screens illustrate the value of this system.

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“…A unidirectional design of the gene-trap cassette overcomes this problem and has facilitated the identification of essential genes in human cells as well as genotype-specific gene requirements by using the distribution of sense and antisense orientations as a readout for gene essentiality ( Wang et al, 2015b ; Blomen et al, 2015 ; Haarhuis et al, 2017 ). Haploid screens have furthermore been employed to study genotypes which sensitize cells to chemical compounds ( Pettitt et al, 2017 ). For instance, we have recently found that loss of the tumor suppressor FBXW7 sensitize cells to Vinca alkaloids.…”

Section: Forward Genetic Screens To Improve Our Understanding Of Drugmentioning

confidence: 99%

New tools for old drugs: Functional genetic screens to optimize current chemotherapy

Gerhards

Rottenberg

2018

Drug Resistance Updates

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Despite substantial advances in the treatment of various cancers, many patients still receive anti-cancer therapies that hardly eradicate tumor cells but inflict considerable side effects. To provide the best treatment regimen for an individual patient, a major goal in molecular oncology is to identify predictive markers for a personalized therapeutic strategy. Regarding novel targeted anti-cancer therapies, there are usually good markers available. Unfortunately, however, targeted therapies alone often result in rather short remissions and little cytotoxic effect on the cancer cells. Therefore, classical chemotherapy with frequent long remissions, cures, and a clear effect on cancer cell eradication remains a corner stone in current anti-cancer therapy. Reliable biomarkers which predict the response of tumors to classical chemotherapy are rare, in contrast to the situation for targeted therapy. For the bulk of cytotoxic therapeutic agents, including DNA-damaging drugs, drugs targeting microtubules or antimetabolites, there are still no reliable biomarkers used in the clinic to predict tumor response. To make progress in this direction, meticulous studies of classical chemotherapeutic drug action and resistance mechanisms are required. For this purpose, novel functional screening technologies have emerged as successful technologies to study chemotherapeutic drug response in a variety of models. They allow a systematic analysis of genetic contributions to a drug-responsive or −sensitive phenotype and facilitate a better understanding of the mode of action of these drugs. These functional genomic approaches are not only useful for the development of novel targeted anti-cancer drugs but may also guide the use of classical chemotherapeutic drugs by deciphering novel mechanisms influencing a tumor’s drug response. Moreover, due to the advances of 3D organoid cultures from patient tumors and in vivo screens in mice, these genetic screens can be applied using conditions that are more representative of the clinical setting. Patient-derived 3D organoid lines furthermore allow the characterization of the “essentialome”, the specific set of genes required for survival of these cells, of an individual tumor, which could be monitored over the course of treatment and help understanding how drug resistance evolves in clinical tumors. Thus, we expect that these functional screens will enable the discovery of novel cancer-specific vulnerabilities, and through clinical validation, move the field of predictive biomarkers forward. This review focuses on novel advanced techniques to decipher the interplay between genetic alterations and drug response.

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Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells

Cited by 15 publications

References 31 publications

Transposons As Tools for Functional Genomics in Vertebrate Models

Transposons As Tools for Functional Genomics in Vertebrate Models

Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens

New tools for old drugs: Functional genetic screens to optimize current chemotherapy

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