Efficient transposition of Tol2 in the mouse germline

Abstract

Insertional mutagenesis screens play an integral part in the annotating of functional data for all sequenced genes in the postgenomic era. Chemical mutagenesis screens are highly efficient but identifying the causative gene can be a laborious task. Other mutagenesis platforms, such as transposable elements, have been successfully applied for insertional mutagenesis screens in both the mouse and rat. However, relatively low transposition efficiency has hampered their use as a high-throughput forward genetic mutagenesis screen. Here we report the first evidence of germline activity in the mouse using a naturally active DNA transposon derived from the medaka fish called Tol2, as an alternative system for high-throughput forward genetic mutagenesis screening tool.

Figure 1.—

Tol2 transposon system in the mouse germline. (a) Comparative study between the endogenous and codon-optimized versions of Tol2. Colony-resistant assay in HeLa cells was used to compare between the transposition activities of both versions of Tol2. A Tol2 transposon vector carrying a Zeocin resistance gene was cotransfected with either endogenous or codon-optimized versions of Tol2 under the control of the cytomegalovirus (CMV) promoter at 500 ng (light shading) and 50 ng (dark shading) concentrations. Using the number of colony-resistant cells as an indication of transposition activity, the codon-optimized version of Tol2 was found to be approximately twofold more active than the endogenous version at both concentrations. CMV-GFP was used as a negative control. Data are the mean ± standard deviation of three individual experiments. (b) Components of the Tol2 transposon and codon-optimized transposase vectors. The pminiTol2 flanking arms (inverted arrows) were used to flank the DNA cassette composed of the internal gene-trap/GFP-reporter components of the transposon vector. The gene-trap component consists of a splice acceptor (SA) fused to the polyadenylation signal (polyA). The GFP-reporter component consists of the cytomegalovirus early enhancer/chicken beta-actin (CAG) promoter driving the GFP gene, followed by the internal ribosome entry site (IRES) and splice donor (SD). The presence of the IRES helps prevent nonsense-mediated mRNA decay. The entire DNA cassette is flanked by loxP sites (arrowheads). The codon-optimized Tol2 (iTol2) was placed under the control of the CAG promoter. (c) Breeding strategy for generating Tol2 mutant mice. Singly transgenic mice for Tol2 transposase and transposon vector were generated separately. Interbreeding the two singly transgenic mice generated a doubly transgenic mouse carrying both components (seed mouse). The doubly transgenic mouse was bred with wild-type FVB/N females to generate many mutant progeny. Tol2 transposase binds to the flanking ends of the Tol2 transposon containing the 12-bp inverted repeats (arrowheads), excises a single copy of the transposon vector from the original donor concatemer (consisting of many copies of the transposon vector) and reintegrates the transposon vector into a different locus. If the transposon is inserted into a transcribed gene in the correct orientation, a GFP signal is produced by the GFP-reporter/poly(A)-trap system. However, if the transposon is inserted outside of a gene or in an incorrect orientation, no GFP signal is produced. This detection allows for the rapid and noninvasive screen for Tol2 mutant mice. (d) PCR genotyping of Tol2 mutant lines. Three mutant lines were selected for PCR genotyping after determining the transposon insertion site using linker-mediated PCR. MW, 100-bp molecular standard; FVB, wild-type FVB/N genomic DNA; P1/P3/P12, Tol2 mutant line genomic DNA; DDW, sterile double-distilled water used as negative control; arrowhead, wild-type allele; open arrowhead, mutant allele. (e) Southern analysis of Tol2 mutant mice. Southern analysis demonstrated new transposition events in GFP⁺ Tol2 mutant mice (average of 3 insertion sites per GFP⁺ mouse). Seed, original seed mouse (doubly transgenic mouse carrying both Tol2 transposon and codon-optimized transposase); P, Tol2 mutant mice; B6, C57BL/6 tail genomic DNA used as negative control; donor, donor concatemer consisting of multiple transposon vector copies; arrowhead, the donor concatemer detected by the GFP probe; open arrowheads, new transposition events occurring in the Tol2 mutant mouse germline. (f) Tol2 displays local hopping phenomenon in the germline. By mapping 55 insertion sites on the putative donor chromosome, the putative donor site was mapped approximately to the 82 Mb locus on chromosome 4C3 due to the clustering of insertion sites around this site. Arrowhead, putative donor site on chromosome 4C3. (g) Confirming the location of the Tol2 transposon vector donor concatemer on chromosome 4 by FISH analysis using splenocytes from a donor-positive transgenic mouse. Tol2 transposon vector donor concatemer, located at band 4C3 (indicated by solid arrowhead) and the chromosome BACs, located at 4D2.2 (indicated by open arrowheads).

Figure 2.—

Fusion Tol2 transposon vector-endogenous transcripts in mutant mouse lines. (a) Fusion transposon vector-endogenous transcripts were detected using 3′ RACE in mutant mouse lines (P13, P31, and P82) (arrowheads denote fusion transcripts). Intact RNA was confirmed by RT–PCR for the beta-actin (Actb) gene. MW, 100-bp molecular standard; RT (+), reverse transcriptase added in the first strand cDNA synthesis reaction; RT (–), reverse transcriptase excluded in the first strand cDNA synthesis reaction. (b) Diagrammatic representation of fusion transcripts determined by 3′ RACE showing the detection of known and possible novel transcripts using the Tol2 transposon system. P31 fusion transcript, transposon vector (arrowhead) inserted in intron 1 of Genscan (chr4.1455). Putative exons shown as black (detected in P31 mutant line only) and gray (detected in both P13 and P31 mutant lines) bars. Dotted arrow indicates the orientation of the endogenous gene. P82 fusion transcript 1, transposon vector inserted into a gene-free region. Putative exons (black bars) detected suggesting the presence of a novel transcript. P82 fusion transcript 2, transposon vector (arrowhead) inserted into intron 5 of Genscan (chr4_806.1). Both novel and known transcripts (EST BB659592) were detected (black bars). All transposon vector insertion sites for all mutant lines were determined by LM-PCR. Fusion sequences for the Tol2 transposon vector Hprt splice donor (lowercase) and mouse endogenous transcripts (uppercase) are shown. Ex, putative exon.