Transposon-mediated genetic screening and quantitative insertion site sequencing for cancer gene discovery in mice

Developers

Mathias J. Friedrich, Lena Rad, Allan Bradley, Roland Rad, etc.

Description of the technology

This is technology of transposon-mediated forward genetics screening in mice, which has become now 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.

The authors give a complete characteristics of 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. A method for semiquantitative transposon insertion site sequencing (QiSeq) has been also developed. 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. Multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Technology also describes principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution. Aging mice until tumors arise is one of solved problems of this method. In this case, monitoring must be adapted to the expected tumor entity and the speed of onset of expected symptoms.

Practical application

The technology is applicable for experimental study of genetic mechanisms of tumors and discovery of cancer genes. The method is also useful for cancer gene network analysis and understanding of tumor evolution stages.

Among advantage of the technology is that it takes into account simultaneously developing ageing processes, which often are similar to some processes of tumor development.

Laboratories

• The Wellcome Trust Sanger Institute, Genome Campus, Hinxton/Cambridge (UK)
• Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich (Germany)
• German Cancer Research Center (DKFZ), Heidelberg, & German Cancer Consortium (DKTK), Heidelberg (Germany)
• Instituto de Medicina Oncológica y Molecular de Asturias (IMOMA), Oviedo (Spain)

Links

Publications

• Friedrich, M.J. et al. «Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice." 12 Nature Protocols (2017): 289–309.
• Rad, R. et al. «A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer." 47 Nat. Genet. (2015): 47–56.
• Rad, R. et al. «A genetic progression model of BrafV600E-induced intestinal tumorigenesis reveals targets for therapeutic intervention." 24 Cancer Cell (2013): 15–29.