Characterization of Sleeping Beauty transposition and its application to genetic screening in mice

Abstract

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.

FIG. 1.

Determination of SB transposition sites in the mouse germ line. (A) Overview of the breeding scheme used to generate mutant mice. grey arrows, IR and DR of the transposon that is the recognition sequence of the SB transposase. GFP was used as a marker to monitor transposition events; other elements located between IRs and DRs are not shown for simplicity. Two kinds of singly positive mice are generated; one carries the transposon vector, and the other expresses the SB transposase. Doubly transgenic mice (seed mice) are obtained as a result of breeding of the singly positive mice and are mated with wild-type mice. Mutant mice are generated as a result of a transposition event in the germ lines of seed mice. (B) Strategy used to study transposition sites in the sperm of seed mice. (C and D) Ligation-mediated PCR to determine independent transposition sites from sperm DNA. (C) PCR procedure; (D) amplification of one copy of a transposition site per reaction. RS, restriction site; filled boxes, linker DNAs; Rx, reaction. The marker (lane M) is a 100-bp DNA ladder.

FIG. 2.

Distribution of SB transposition sites in the mouse germ line. (A) Distribution of transposition sites from a single copy of the SB transposon at the DST. Twelve transposition sites described previously (10) were analyzed again by using the Celera Genomics database, and three transposition sites located within 200 kb of the DST are shown. CEN, centromeric region; TEL, telomeric region. The DST is depicted by a filled circle. Arrows indicate transposition sites of loci 1835, 1818, and 1680 (Table 1). (B and C) Distribution of transposition sites from the seed mice of line A (B) and line B (C) according to the Ensembl mouse genome database. The DST was mapped by FISH to chromosome 14 B distal-C1 proximal in line A (data not shown) and to chromosome 3 H1-H2 in line B as reported previously (16). Chromosomes bearing DSTs were divided into 200-kb intervals, and the number of transposon insertions per interval was plotted. The 20-Mb regions around the cluster of insertions are shown magnified as well, together with a 3-Mb scale of black and white boxes. Note that although some transposition sites are clustered, most of them were mapped to different locations at the nucleotide level (see Table 2).

FIG. 3.

Distribution of transposon insertion sites at known or predicted genes. Transposon insertion sites that were mapped between 10 kb upstream and 10 kb downstream of the transcription units in Tables 1 and 2 are shown. They are classified into two patterns: single insertion per gene (A) and multiple insertions in a single gene (B). When a gene is registered in both the Ensembl and Celera databases (Tables 1 and 2), the structure of the gene and the corresponding accession number are shown according to the Ensembl database. Boxes, exons; arrows, transposon insertion sites. The orientation of genes is from left to right. Scale bars for each of the genes are shown.

FIG. 3.

Distribution of transposon insertion sites at known or predicted genes. Transposon insertion sites that were mapped between 10 kb upstream and 10 kb downstream of the transcription units in Tables 1 and 2 are shown. They are classified into two patterns: single insertion per gene (A) and multiple insertions in a single gene (B). When a gene is registered in both the Ensembl and Celera databases (Tables 1 and 2), the structure of the gene and the corresponding accession number are shown according to the Ensembl database. Boxes, exons; arrows, transposon insertion sites. The orientation of genes is from left to right. Scale bars for each of the genes are shown.

FIG. 3.

Distribution of transposon insertion sites at known or predicted genes. Transposon insertion sites that were mapped between 10 kb upstream and 10 kb downstream of the transcription units in Tables 1 and 2 are shown. They are classified into two patterns: single insertion per gene (A) and multiple insertions in a single gene (B). When a gene is registered in both the Ensembl and Celera databases (Tables 1 and 2), the structure of the gene and the corresponding accession number are shown according to the Ensembl database. Boxes, exons; arrows, transposon insertion sites. The orientation of genes is from left to right. Scale bars for each of the genes are shown.

FIG. 3.

Distribution of transposon insertion sites at known or predicted genes. Transposon insertion sites that were mapped between 10 kb upstream and 10 kb downstream of the transcription units in Tables 1 and 2 are shown. They are classified into two patterns: single insertion per gene (A) and multiple insertions in a single gene (B). When a gene is registered in both the Ensembl and Celera databases (Tables 1 and 2), the structure of the gene and the corresponding accession number are shown according to the Ensembl database. Boxes, exons; arrows, transposon insertion sites. The orientation of genes is from left to right. Scale bars for each of the genes are shown.

FIG. 4.

Complexity of transposition sites within the germ lines of seed mice. (A) Nested PCR primers for the detection of an individual transposition. Primers p1 and p2 are for the first reaction, and primers p3 and p4 are for the second reaction. The region represented by a black bar was isolated by PCR as shown in Fig. 1C, and this fragment was used as a template in lanes 1 to 10 of panels B and C. (B and C) Estimation of the complexity of transposition sites in line B (B) and line A (C). The transposition sites being studied are indicated on the left of each panel. Lanes 1 to 10 show the PCR sensitivity. Lanes 11 to 14 show that each transposition site was detected only in the parental mouse. Lanes 15 to 22 show the amount of testicular DNA in which the target molecule exists. The marker (lanes M) is a 100-bp DNA ladder. Rx, reaction. (D) Schematic diagram of complexity of transposition sites in seed mice. Overlapping circles depicting complexity indicate that some transposition sites are clustered close to the donor site in the same mouse strain.

FIG. 5.

Screening for mutant mice generated by the gene trap strategy. (A) Strategy to identify transposition events by using the previously reported old version of the SB vector (16) (left) (also see Fig. 1A) and the new SB trap vector used in the present study (right). pA, poly(A) addition signal; SD, splice donor; filled boxes, exons. (B) Outline of the gene trap scheme. In this example, the transposon vector is inserted into intron 2 of an endogenous gene. Transcription of the endogenous gene results in a chimeric transcript of the endogenous transcript and vector-derived sequences. As a result, translation from the endogenous gene is disrupted and β-galactosidase is expressed, reflecting the expression pattern of the endogenous gene. Transcription by the ubiquitously active CAG promoter generates a chimeric transcript of the GFP sequence and the endogenous transcript, resulting in ubiquitous expression of GFP. White boxes, untranslated regions of exons; black boxes, translated regions of exons. (C) Screening for mutant mice performed by GFP expression. Newborn mice were examined by fluorescence stereomicroscopy. The left panel is a bright-field image, and the right panel is a dark-field fluorescent image taken with GFP filters. The mouse at the top is GFP positive (and therefore presumably a mutant), and the one at the bottom is GFP negative. (D) Multiple insertions into the neurexin 3 gene by local transposition. The neurexin 3 gene contains two promoters, one transcribing alpha-neurexin 3 and the other transcribing beta-neurexin 3, and is located in the vicinity of the DST. Thick arrows indicate the locations of trapped sites, and those with asterisks indicate that the vector-derived SD site was spliced to exons. The orientation of the transposon insertion is shown by thin arrows.

FIG.6.

Distribution of trapped sites at known or predicted genes. From the trapped sites that were mapped at the genes shown in Table 3, 18 sites with transposon insertions in the same orientation relative to the trapped genes are shown. Insertions into neurexin 3 genes (TM115 and TM189) are shown in Fig. 5D and therefore are not shown here. Splicing patterns revealed by 3′ RACE are shown at the top. In addition to the predicted splicing between the vector-derived SD site and genomic sequences (type I transcript), we occasionally observed unexpected splicing between the SD site and a cryptic SA site within the trap vector (type II transcript). Since the type II transcript contains the junction of the transposon sequence and the genomic sequence, we sequenced it to determine the transposon insertion site (for TM29, TM42, TM67, and TM73). When a type II transcript was not observed, transposon insertion sites were determined either by ligation-mediated PCR (7, 10) (for TM21, TM22, TM75, TM90, TM117, and TM195) or by PCR between transposon-specific primers (T/BAL and T/DR) (18) and reverse primers that were designed at or upstream of the trapped site (TM88). TM numbers correspond to the mouse identification numbers in Table 3. When a gene is registered in both the Ensembl and Celera databases (Table 3), the structure of the gene and the corresponding accession number are presented according to the Ensembl database. Black arrows indicate the locations of trapped sites, and asterisks indicate that the vector-derived SD site was spliced to known or predicted exons. White arrows indicate transposon insertion sites. The orientation of genes is from left to right. Scale bars for each of the genes are shown. For TM67, see the legend for Fig. 7E.

FIG. 7.

Generation and analysis of mutant mice. (A to C) X-Gal staining of an 11.5-dpc embryo from TM75 (A), an adult testis from TM90 (B), and an 11.5-dpc embryo from TM67 (C). (D) lacZ gene induction by dexamethasone in the thymuses of TM88 mice. Heterozygous mice (10 days of age, 6.5 g) were injected intraperitoneally with 200 μg of dexamethasone, and the thymus was stained with X-Gal 16 h after injection. (E and F) Disruption of gene expression in homozygous TM67 mutant mice (E) and TM88 mutant mice (F). p1 and p3 were used to detect the wild-type (wt) transcripts of the mutated genes, and p2 and p3 were used to detect splicing events occurring between the transposon vector and a downstream exon. We found an uncharacterized EST (National Center for Biotechnology Information accession no. 11506469) derived from the upstream region of the mtsh2 gene and incorporated it in the gene structure presented in panel E. (G) TM117 heterozygous mice were established after segregating the DST during breeding and were intercrossed. Blastocysts were isolated and observed for 3 days, followed by genotyping with PCR. Hatching was impaired in homozygous embryos.