PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice

Abstract

Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.

Figures

Fig. 1. Mouse lines carrying the genetic components of the transposon systems

(A) RosaPB and RosaSB knock-in mice express PiggyBac or Sleeping-Beauty transposase under control of the constitutively active Rosa26 promoter. (B) Transposon design and transposon mouse lines. Left: Three transposon constructs were designed, which differ in their promoter/enhancer. All three transposons have PB as well as SB inverted terminal repeats (ITRs) and can therefore be mobilized with both transposases. Right: Three types of transposon mouse lines (ATP1, ATP2 and ATP3) were generated using these constructs. A total of 19 ATP lines were developed and are listed in the tables on the right. For each line the chromosome harbouring the transposon donor locus and the transposon copy number in the donor concatemer are indicated. CβASA, Carp β-actin splice acceptor; En2SA, Engrailed-2 exon-2 splice acceptor; SD, Foxf2 exon-1 splice donor; pA, bidirectional SV40 polyadenylation signal; CAG, CMV enhancer and chicken beta-actin promoter; MSCV, murine stem cell virus LTR; PGK, phosphoglycerate-kinase promoter;

Fig. 2. Embryonic lethality, tumor spectrum and latency in different ATP mouse lines upon PiggyBac mobilization

(A) Embryonic lethality in offspring from RosaPB;ATP intercrosses. 14 ATP mouse lines were used. Each line’s transposon copy number is indicated below the graph. The number of mice born in each colony is shown above the graph. Bars represent double transgenic progeny as a percentage of expected. (B; C; E) Tumor spectrum and latency in different ATP mouse lines upon PB transposon mobilization. The percentage of mice with hematopoietic or solid tumors is indicated for RosaPB;ATP2 (B), RosaPB;ATP1 (C) and RosaPB/ATP3 mice (E). Kaplan-Meier survival curves are shown for indicated genotypes. (D) CAG promoter-driven DsRed expression in mouse tissues. Left columns: red fluorescence detection in organs from wild-type (WT) and hprtCAG-DsRed (R) male mice. H, heart; M, skeletal muscle; K, kidney; B, brain; T, testicle; IN, small intestine; L, lung; S, spleen. Right columns: Western blot analysis of the same organs using a DsRed-specific antibody (r) or control antibodies (t, alpha-tubulin; a, beta-actin).

Fig. 3. PB integration pattern in tumors from different ATP2 mouse lines

(A) Analysis of tumors from RosaPB;ATP2-S1 mice (50 tumors; 4379 insertions). The lower graph compares the percentage of transposon insertions to the size and TTAA frequency on individual chromosomes. The donor chromosome and locus were identified by FISH and are indicated by blue arrows. The upper graph shows the distribution of insertions on Chr17 at a 1 Mb resolution. The number of expected (exp) and observed insertions outside (odl) and inside (idl) a 3MB interval surrounding the donor locus are indicated. (B) Results from RosaPB;ATP2-S2 mice (7 tumors; 594 insertions; donor Chr12), and RosaPB;ATP2-H32 mice (6 tumors; 618 insertions; donor Chr2) are presented accordingly.

Figure 4. Identification of common integration sites in PiggyBac-induced hematopoietic tumors

(A) Pie chart representing all CIS identified: Some were reported in earlier screens using retroviruses (RV; 36%), SB (4%) or both (18%). 42% have not been identified in earlier screens. (B) Insertion pattern in known oncogenes and tumor suppressor genes. Arrows indicate individual insertions and their orientation (red/blue, sense- or antisense-orientation of the transposon’s promoter to genes, respectively). (C) Promoter insertions in Spic in 9 tumors. MSCV-Spic fusion transcripts were detected by RT-PCR and sequencing. Spic expression was analyzed by qRT-PCR and normalized to Gapdh expression. Tumors with Spic integrations are myeloid leukemias, as shown by immunohistochemical myeloperoxidase staining (left, spleen; right lymph node from one mouse). Scale bars, 25μm. ***p<0.001