Journal
NATURE PROTOCOLS
Volume 12, Issue 2, Pages 289-309Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.164
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Funding
- Wellcome Trust
- German Cancer Consortium (DKTK)
- German Research Council [SFB1243]
- Helmholtz Association (PCCC Alliance)
- Medical Research Council [MC_PC_12009] Funding Source: researchfish
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Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction-digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.
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