Transposon-mediated mutagenesis in somatic cells: identification of transposon-genomic DNA junctions

Abstract

Understanding the genetic basis for tumor formation is crucial for treating cancer. Forward genetic screens using insertional mutagenesis technologies have identified many important tumor suppressor genes and oncogenes in mouse models of human cancer. Traditionally, retroviruses have been used for this purpose, allowing the identification of genes that can cause various forms of leukemia or lymphoma with murine leukemia viruses or mammary cancer with mouse mammary tumor viruses. Recently, the Sleeping Beauty transposon system has emerged as a tool for cancer gene discovery in mouse models of human cancer. Transposons mobilized in the mouse soma can insertionally mutate cancer genes, and the transposon itself serves as a molecular "tag," which facilitates candidate cancer gene identification. We provide an overview of some general issues related to use of Sleeping Beauty for cancer genetic studies and present here the polymerase chain reaction-based method for cloning transposon-tagged sequences from tumors.

Fig. 1

T2/onc. T2/onc has SA/polyadenylation (pA) sequences in both orientations to generate loss-of-function mutations in genes in which it lands. Between the two SA are sequences from the long terminal repeat of the MSCV LTR, which contains promoter/enhancer elements to promote overexpression of genes near which it lands. Also present is a splice donor (SD) so that transcripts initiated in the MSCV LTR can splice into downstream endogenous exons.

Fig. 2

Outline of linker-mediated PCR for cloning proviral- or transposon-genomic DNA junctions. Linker-mediated PCR involves the generation of transposon (or proviral)-genomic DNA fragments by restriction digest of tumor genomic DNA. Linkers with overhangs complementary to the restriction fragments used are then ligated onto the genomic DNA. Two rounds of PCR with linker and IRDR (or proviral) specific primers are used to amplify the transposon-genomic DNA junction.

Fig. 3

A typical linker-mediated PCR result on leukemias induced by T2/onc insertional mutagenesis. Most successful linker-mediated PCR reactions produce a “smear” of PCR products, although some individual bands can be discerned.