Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec 16;105(50):19904-9.
doi: 10.1073/pnas.0807785105. Epub 2008 Dec 8.

A DNA transposon-based approach to validate oncogenic mutations in the mouse

Qin Su  1 , Haydn M Prosser, Lia S Campos, Mariaestela Ortiz, Takuro Nakamura, Madhuri Warren, Adam J Dupuy, Nancy A Jenkins, Neal G Copeland, Allan Bradley, Pentao Liu
Affiliations

A DNA transposon-based approach to validate oncogenic mutations in the mouse

Qin Su et al. Proc Natl Acad Sci U S A. .

Abstract

Large-scale cancer genome projects will soon be able to sequence many cancer genomes to comprehensively identify genetic changes in human cancer. Genome-wide association studies have also identified putative cancer associated loci. Functional validation of these genetic mutations in vivo is becoming a challenge. We describe here a DNA transposon-based platform that permits us to explore the oncogenic potential of genetic mutations in the mouse. Briefly, promoter-less human cancer gene cDNAs were first cloned into Sleeping Beauty (SB) transposons. DNA transposition in the mouse that carried both the transposons and the SB transposase made it possible for the cDNAs to be expressed from an appropriate endogenous genomic locus and in the relevant cell types for tumor development. Consequently, these mice developed a broad spectrum of tumors at very early postnatal stages. This technology thus complements the large-scale cancer genome projects.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Construction and targeting of the Onco-Array into the mouse Hprt locus. (A) Diagram of the basic strategy used in this study. A promoter-less cDNA is cloned into the SB transposon (Onco-vector) that is subsequently engineered into a genomic locus. The cDNA can be expressed once the SB transposon jumps out of the original locus and reintegrates into a new locus in the genome. LTR and RTR, left and right terminal inverted repeats of the Sleeping Beauty transposon; SA, adenovirus splicing acceptor; pA, polyadenylation signal sequence of bovine growth hormone gene (bGHpA). The cDNA has the Kozak sequence at the 5′ end of the coding sequence. (B) Targeting the Onco-Array into the Hprt locus via recombination mediated cassette exchange (RMCE). The nine SB transposon units (indicated by the cDNAs that they carry) were cloned into a vector that had loxP and lox511 sites for RMCE and a PGKNeobpA cassette for positive selection of the integration events. To avoid transcription from the Hprt promoter, the cDNAs in the Onco-Array are in the opposite orientation with the Hprt transcription direction. The multiple pA sequences were designed to minimize any potential readthrough transcription activities. PGKPurodeltaTKpA is the selection cassette for inserting the loxP and lox511 to the Hprt locus.
Fig. 2.
Fig. 2.
Development of benign and malignant tumors in the double transgenic mice at early ages. (A and B) Angiosarcoma and abnormal brain vascularization and hemorrhage in mouse PLND15.x1. Note that the endothelial cells are plump and there is brisk mitotic activity. (C) Angiosarcoma in mouse PLND4.2f. (D and E) Medulloblastoma in the inferior surface of the telencephalon of mouse PLND8.y. This tumor expressed MYC, a hallmark of medulloblastoma, as seen in (F). (G and H) A rhabdomyosarcoma in mouse PLND25.1 positively stained with a KRAS G12D-specific antibody (i). (J) A well-differentiated squamous cell carcinoma of the skin in mouse PLND4.2f. (K) Lymphoid infiltration or small cell carcinoma in the spleen of mouse PLND15.x1. (L) A non-small cell lung tumor with morphological features of bronchoalveolar carcinoma in mouse PLND21. (Magnification: A, D, and K ×100; B, C, E–G, I, and L ×200; H and J ×400.)
Fig. 3.
Fig. 3.
SB transposon integration sites in the tumors and examination of tumor clonality. (A) Genomic DNA samples were digested with XbaI and probed with a DNA fragment corresponding to the SB transposon LTR plus the SA sequence in Onco-vector (see diagram at bottom of figure). The arrow points to the 500 bp Onco-Array germline band, whereas the tumors had multiple independent SB transposon integration sites. (B) The same Southern blot was probed with a DNA fragment from TcrJβ1 that detected the 3.0 kb germline band. Two of the three tumors (lane 3 and lane 5) had prominent Tcrβ rearrangement bands, confirming the clonality of these tumors. Lane 1: wild-type mouse liver; lane 2: PLND mouse (no SB transposase) tail; lane 3: T-cell leukemia from recipient mouse 114106; lane 4: T-cell leukemia from recipient mouse 109038; and lane 5: T-cell leukemia from recipient mouse 109659.
Fig. 4.
Fig. 4.
Identification of the SB transposon integration sites. (A) Diagram of splinkerette PCR used to identify the SB transposon integration sites in the tumors. Genomic DNA is first digested with Sau3A and subsequently ligated to the splinkerette linker. The genomic DNA-transposon junction DNA fragments are amplified in two rounds of PCR. (B) Integration of the SB transposon carrying the human mutant KRAS2B in the intron 1 of the Stag2 locus. (C) The fusion transcript between the Stag2 exon 1 and the KRAS2B in the transposon was amplified in RT-PCR and sequence verified as shown in (D).
Fig. 5.
Fig. 5.
Only the mutant forms of KRAS and BRAF were expressed in the tumors. (A) RT-PCR analysis of the tumor cells in the spleen of mouse PLND4.2f showed that KRAS2A, KRAS2B, BRAF, and MYC were all expressed in this sample. (B) Sequencing of the RT-PCR products showed that the expressed cDNAs of KRAS2A, KRAS2B, and BRAF were predominantly the mutant forms.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. - PubMed
    1. Futreal PA, et al. A census of human cancer genes. Nat Rev Cancer. 2004;4:177–183. - PMC - PubMed
    1. Greenman C, et al. Patterns of somatic mutation in human cancer genomes. Nature. 2007;446:153–158. - PMC - PubMed
    1. Wood LD, et al. The genomic landscapes of human breast and colorectal cancers. Science. 2007;318:1108–1113. - PubMed
    1. Sjoblom T, et al. The consensus coding sequences of human breast and colorectal cancers. Science. 2006;314:268–274. - PubMed

Publication types

LinkOut - more resources