Identification of <em>Sleeping Beauty</em> Transposon Insertions in Solid Tumors using Linker-mediated PCR

Abstract

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer 1 . Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation . The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR ( . Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain-and loss-of-function mutations 6 . The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1). Video LinkThe video component of this article can be found at

Sleeping Beauty Insertional Mutagenesis in Mice Identifies Drivers of Steatosis-Associated Hepatic Tumors

“…Transposon-genomic DNA junctions were amplified from genomic DNA isolated from SB-induced liver tumors by linker-mediated PCR as described(17) and sequenced on the Illumina MiSeq with 150 cycle paired-end reads. Non-redundant insertions were calculated using TAPDANCE; to limit analysis to trunk drivers, only reads over 1/10000th of the total reads for the tumor were included.…”

Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery

“…For example, most SB screens use ligation-mediated polymerase chain reaction (LM-PCR) based recovery of transposon insertion sites from tumor DNA [25]. However, almost all screens to date use restriction enzymes as a first step in the LM-PCR protocol.…”