Design of a Screen to Mutagenize Mouse Chromosome 11

Transposase-negative offspring of seed mice that inherited the transposon donor site by Southern blot (Figure 1B) were designated with a pedigree letter and entered as first-generation animals into a three-generation breeding scheme (Figure 1C). We took advantage of the Inv(11)8Brd^Trp53–Wnt3 strain of mice to preferentially homozygose local transposon-induced mutations in Chromosome 11 between Trp53 and Wnt3. Carriers of the balancer chromosome are identified by expression of an Agouti transgene incorporated into one breakpoint of the engineered inversion [11]. Additionally, animals inheriting two copies of the balancer chromosome die in utero because one inversion breakpoint is in the essential Wnt3 gene [11].

The results of the ENU screen [11,12] gave insight into the phenotypes we might expect to recover, and importantly, might be rare to discover in a screen. Recessive lethal (63%), neurological (11%), skeletal (6%), and growth/size defects (6%) made up the majority of recovered phenotypes in that study [11]. Anticipating recovery of a similar range of phenotypes, a comparable phenotype screen was designed to recover mutations resulting in growth/size, neuromuscular, or behavioral phenotypes and to identify craniofacial or limb defects (see Materials and Methods). Two control pedigrees, generated in the absence of the transposase strain, were randomly introduced into the screen and were found to be phenotypically normal. Thirty-eight pedigrees were screened using this strategy, resulting in one visible, one behavioral, and 21 recessive, prenatal lethal phenotypes (Table 1).

Table 1

Pedigree Phenotype and Cummulative Insertion Data

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Local Transposition and Gene Mutation on Mouse Chromosome 11

Transposon insertions in the genomes of mutant mice were characterized using previously described methods [6] to measure progress on saturating this gene-dense region with mutations and to catalog any gene disruptions. The NCBI m34 build (May 2005 freeze) was used to identify the genomic position of 175 unique T2/GT3/tTA insertion sites using the BLAST function of the ENSEMBL genome browser http://www.ensembl.org/Mus_musculus/blastview. A full description of the genomic position for the local and genome-wide insertions can be found in Table S1. As predicted, 104 insertions (59%) were localized to mouse Chromosome 11 and, of those, 75 mapped to the balanced region between Trp53 and Wnt3 (Figure 2). In total, 67% (50/75) of insertions within the balanced region were inserted into 38 different known or predicted genes (Table S1).

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Figure 2

Distribution of Chromosome 11 Insertions

The insertions over the entire Chromosome 11 and the gene-dense, balanced region between Trp53 and Wnt3 are shown as a histogram over the ENSEMBL ContigView (http://feb2006.archive.ensembl.org/Mus_musculus/contigview?region=11&vc_start=69.3M&vc_end=103.6M&h=11). The number of insertions over the whole chromosome is shown in 1-Mbp bins, while the balanced region is shown in 100-kb bins.

Dominant Polysyndactyly and Recessive Hyperactivity Phenotypes of Mutant Pedigrees

Dominant polysyndactyly is evident in carriers of the mutagenized Chromosome 11 in pedigree BM (Figure 3A). These mice demonstrate duplication and often fusion of the anterior digit on the fore and hind limbs. The open field test, performed in the SHIRPA arena (see Materials and Methods), identified a recessive hyperactivity phenotype as homozygous BG animals traveled through more squares when compared to their control siblings (Figure 3B).

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Figure 3

Visible and Behavioral Phenotypes as a Result of Transposon Mutation

(A) Dominant polydactyly (extra digits) or polysyndactyly (extra, fused digits) is evident in the fore (top) and hind limbs (bottom) of animals in pedigree BM.

(B) A recessive hyperactive phenotype is measured by the number of squares visited in the SHIRPA arena (see Materials and Methods) in homozygous animals in the viable pedigree BG (p = 0.0113 by unpaired t-test).

Candidate gene insertions were identified in the mutagenized Chromosome 11 of pedigrees BM and BG. Heterozygous pedigree BM mice harbor an insertion (03A-0204, Table S1) in a pioneer gene (UniProt/TrEMBL ID Q5SRA9). This gene is highly conserved throughout all major animal phyla, yet no clues as to its function have been reported in other model organisms. Homozygous pedigree BG animals display the hyperactive phenotype and harbor an insertion (03A-0355, Table S1) in a member of a family of genes known to be expressed in the nervous system and that have been associated with neurological disorders. We are currently attempting to correlate these candidate single-gene disruptions on Chromosome 11 with phenotypes. However, subsequent analysis of the recessive prenatal lethal phenotypes led us to discover that other types of mutations were present on Chromosome 11 in these and other pedigrees, as is described below. Thus, it is possible that transposon insertions are not responsible for these viable phenotypes.

Recessive Lethality as a Result of Transposon Mutagenesis of Chromosome 11

As mentioned above, 21 of the 38 pedigrees demonstrated recessive lethality, all of which were prenatal lethal (Table 1). While we anticipated a high rate of recessive lethal phenotypes, this result was in stark contrast to the ENU study where out of 55 (recently updated to 59 [12]) recessive lethal phenotypes isolated, only 30 were characterized as prenatal [11]. Based on the initially reported 55 of 88 balanced ENU-induced mutations resulting in lethal phenotypes [11], this region of the genome may harbor a higher than average density of essential mouse genes. SB transposition-induced lethal pedigrees harbored nearly twice as many potential gene disruptions per pedigree on average than non-lethal pedigrees (1.7 vs. 0.9) (Table 1). One possibility is that the germline mobilization rate is sufficiently high such that the screen would identify lethal mutations at this rate. We addressed this hypothesis by both analyzing candidate gene disruptions and complementation testing between lethal pedigrees.

Candidate gene insertions in lethal pedigrees were examined for their association with lethal phenotypes. Carriers were repeatedly outcrossed until recombinants were obtained separating pedigree M insertion 03A-0033, in the mouse carbonic anhydrase 10 gene (Car10), and pedigree V insertion 03A-0063, in the mouse myosin heavy chain 2 gene (Myh2) (Table S1), from all other insertions and the donor site (unpublished data). Carriers of the isolated insertions were intercrossed to discover any associated recessive phenotypes. Homozygous carriers of either insertion were fertile, healthy mice, and displayed no visible or behavioral abnormalities in our assays, showing that the lethality in these pedigrees was not due to transposon insertions in these genes. These gene trap insertions were subsequently characterized to determine their effect on gene expression. Figure 4A shows the RT-PCR analysis on tissues from wild type, carrier, and homozygous animals for each insertion. Insertion 03A-0033 does not appear to alter expression of Car10, although insertion 03A-0063 ablates expression of Myh2 at the level of transcription. We have previously published that the carp ß-actin splice acceptor in the T2/GT3/tTA does not always function to intercept splicing as designed [8] and is apparently defective in disrupting Car10 expression here. The reverse orientation splice acceptor (Figure 1A), derived from the mouse hypoxanthine phosphoribosyltransferase gene [8], however, is effective in the case of the Myh2 insertion.

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Figure 4

Molecular Analysis of T2/GT3/tTA Gene Disruptions

(A) RT-PCR analysis of wild-type (−/−), hemizygous (−/+), and homozygous (+/+) carriers of insertions 03A-0033 and 03A-0063 are compared. Primers to the housekeeping Gapdh gene were used as an internal control for sample quality.

(B) Immunohistochemical staining of wild-type (wt), carrier, and null soleus muscles for MyHC type IIa (top) and type I (bottom). Samples were co-stained with Anti-Laminin to outline individual fibers.

Myh2 encodes the myosin heavy chain IIa peptide (MyHC-IIa) and is one of three adult fast skeletal muscle myosin genes clustered on mouse Chromosome 11 [14]. Immunohistochemical staining of the soleus muscle from homozygous carriers of insertion 03A-0063 demonstrate lack of MyHC-IIa in muscle fibers, compared to carrier and wild-type sibling controls (Figure 4B). Null mutations in the other two Chromosome-11 fast muscle myosins, IIb [15], and IId [16], are also viable, but they demonstrate severe muscle pathology. It was demonstrated in those studies that neighboring Chromosome-11 MyHC genes can compensate for the loss of expression of one MyHC in mutant mice, though they are not functionally redundant [14]. Figure 4B presents evidence that MyHC genes on other mouse chromosomes can compensate as well since type I MyHC, encoded by the Myh7 gene on mouse Chromosome 14, is upregulated in IIa mutant muscle.

Complementation Testcrosses Reveal Major Complementation Groups

To assess whether any of the lethal pedigrees were allelic, complementation testcrosses using G2 animals were performed. ENU mutagenesis in the same region of the genome revealed 23 complementation groups among 24 lethal pedigree testcrosses [11]. In contrast, most of the pedigrees generated by transposon mutagenesis fell into one of two major complementation groups (designated I and II, Figure 5), suggesting that most of our lethal pedigrees were the result of one of two mutations occurring repeatedly in our screen. Interestingly, pedigrees Z and BL failed to complement all pedigrees except AG, AX, and BC, which complemented all pedigrees tested with the single exception being that AX did not complement pedigree AS. Pedigrees Z and BL, therefore, have mutations that overlap with both major complementation groups, while AS likely has more than one lethal mutation since it fails to complement group I pedigrees and, separately, pedigree AX. Pedigrees AG and BC have unique lethality-inducing mutations. Additionally, pedigrees CQ and CD, generated with the RosaSB11 source of transposase, fell into groups I and II, respectively, suggesting that neither reducing the transposition rate, nor using the catalytically enhanced SB11 transposase [17], can eliminate the incidence of the mutations arising in the two major complementation groups. Having cloned many insertions from these mice, the prevalence of two major complementation groups could not be explained by the repeated disruption of the same essential mouse genes (Table S1). Thus, we investigated whether other types of mutations might occur near the donor site to explain these observations.

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Figure 5

Complementation Testcrosses

For each testcross, heterozygous animals from independent lethal pedigrees (see Table 1) were intercrossed to obtain offspring that inherited both copies of their respective mutagenized chromosomes as detected by PCR genotyping or balancer screening (see Figure 1B). Noncomplementation (−) and complementation (+) divided the lethal pedigrees into at least six complementation groups, with two major groups labeled I and II. Pedigrees highlighted in pink complemented every other pedigree tested except one case, where AX failed to complement AS. Pedigrees in blue failed to complement pedigrees other than AG, AX, and BC (purple).

Microdeletions, Inversions, and Chromosomal Translocations as a Result of Transposition

To determine whether local genomic rearrangements are present near the donor site of mutant chromosomes, FISH was used to probe control and mutant pedigree chromosomes. Several bacterial artificial chromosome (BAC) clones were obtained spanning a 1-Mb region from 89.6–90.6 Mb (according to ENSEMBL m34 build, May 17, 2005 freeze) and flanking the presumptive donor site based on insertion data (Figure 2). Each of five BAC clones (Figure 6A), detected with rhodamine (red), was co-hybridized with a fluorescein-detected (green) transposon probe to metaphase chromosome preparations. Red signals for each probe were present near the green donor site of GT3A control mice, suggesting that none of the regions covered by the probes were deleted in the initial transgenesis (unpublished data).

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Figure 6

Molecular Evidence of Chromosomal Rearrangements after Transposition

(A) Five BAC probes (red bars) were designed to the Chromosome 11 region from approximately 89.6–90.6 Mb (ENSEMBL m34 build, May 17, 2005 freeze). The transposon donor site (green) is presumed to be within the bracketed area based on the accumulation of insertions in this region. Single copies of the transposon were not detectable in this assay. Representative FISH hybridizations to metaphase preparations are shown.

(B) Evidence for deletion of BAC signals 424I8 and 107H16 in pedigree W (white arrows).

(C) Translocation of transposons (white arrowheads) along with distal Chromosome 11 sequence (yellow arrows) in pedigree AG.

(D) Evidence for inversion of a large region of Chromosome 11 is detected by BAC probes 367E18 and 297L3 (red arrows) in pedigree BC, likely involving multiple copies of the transposon.

Hybridization of pedigree W demonstrated a loss of signal from BAC probes 424I8 and 107H16 (Figure 6B, white arrows) when compared to the normal copy of Chromosome 11, suggesting a loss of more than 300 kb of genetic material adjacent to the donor site. Pedigree AG was previously confirmed to be a lethal pedigree because homozygous carriers of an insertion near the concatemer donor site (03A-0071, Table S1) cannot be generated (p <0.01 by χ² analysis, unpublished data). BAC probing revealed a sizeable insertion involving a portion of the concatemer (Figure 6C, white arrowheads), and at least 400 kb of distal Chromosome 11 sequence overlapping probes 403C3 and 107H16 (yellow arrows), into distal Chromosome 5 as determined by spectral karyotyping (unpublished data). Thus, these mice have two normal copies of Chromosome 11, which explains why pedigree AG complements all other pedigrees (Figure 5). This is the second example of insertion of the GT3A concatemer into another chromosome, as we previously reported the insertion of a large portion of the concatemer into Chromosome 4 and subsequent local transposition from the translocated donor site to a nearby gene [8]. A third mutant pedigree revealed a large inversion covering tens of millions of base pairs. Multiple green transposon signals can be observed in pedigree BC, suggesting multiple copies of the gene-trap tTA vector were involved in complex rearrangements since a single copy of the transposon could not be detected by this probe (unpublished data). One rearrangement situated the red signals for probes 297L3 and 367E18 to a more proximal position along Chromosome 11 when compared to the normal copy (Figure 6D, first two panels). Based on the physical distance of the probes, this inversion likely encompasses more than 50 million base pairs.

The representational oligonucleotide micro array (ROMA) technique was developed to identify genome-wide copy number variation in tumor DNA [18]. ROMA analysis of mutant pedigree genomic DNA verified that, although the rest of the genome was unaffected (unpublished data), a reduction in copy number of a portion of mouse Chromosome 11 was seen in pedigree W and an amplification of Chromosome-11 sequences was seen in pedigree AG (Figure 7A). Based on the UCSC mm5 build (May 2004 freeze), pedigree W deletion extends from approximately 89.7–90.1 Mb, a loss of ~400 kb of sequence, while the amplification (due to insertion of sequences from Chromosome 11, Figure 6C) in pedigree AG was a roughly 1.9-Mbp region from ~90.3–92.2 Mb. This insertion is detected as amplification by ROMA because the region of Chromosome 11 that would have shown a deletion was bred away. Thus, pedigree AG animals now have two normal copies of Chromosome 11 in addition to the inserted 1.9-Mbp region into Chromosome 5.

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Figure 7

Defined Genomic Rearrangements Caused by Transposition

(A) Moving average plots of ROMA data of deletion in pedigree W and amplification (due to insertion) in pedigree AG.

(B) Summary of rearrangements as determined by FISH (black bars) or ROMA (blue bars). The green box represents the concatemer, though its position relative to the deletions is speculative. ROMA detects loss (---) of chromosomal material, and defines the minimal overlapping regions for complementation groups 1 (blue box) and 2 (orange box), as well as amplification (+++) of genomic sequences. Where the FISH method was used (black bars) the true extent of each rearrangement could not be determined.

Figure 7B summarizes ROMA and FISH data collected for Chromosome 11 rearrangements from various pedigrees. We propose that the two major complementation groups (Figure 5) can be explained by overlapping deletions that occurred during transposition. Complementation group I, represented by pedigrees AL, BB, AS, V, BM, and CQ, carry overlapping deletions detectable by ROMA (blue bars) or FISH (black bars) in a region of Chromosome 11 immediately distal to the predicted concatemer donor site (Figure 7B). This region harbors the syntaxin binding protein 4 gene, and mouse homologs of a yeast cytochrome C oxidase assembly protein, and of the chicken target of myb1-like 1 (Tom1l1) genes (blue box, Figure 7B). Even though it is a member of complementation group I, pedigree AO did not harbor a deletion detectable by the ROMA technique. This pedigree, rather, harbors multiple transposon insertions, one within the non-complementation region just defined by the pedigrees above, situated in Tom1l1 (insertion 03A-0297, Table S1), and may indicate that Tom1l1 is essential.

In contrast to those distal mutations caused by transposition, pedigrees W, BH, AP, and AY, members of complementation group II, have overlapping deletions which are proximal to the predicted donor site. This region contains the monocyte to macrophage differentiation-associated and hepatic leukemia factor genes (orange box, Figure 7B). ROMA analysis of pedigree AP also identified a tandem duplication of Chromosome-11 sequences distal to the donor site (Figure 7B).

The ~4.3-Mbp deletion in pedigree Z, detected by ROMA, explains why this pedigree fails to complement either group I or group II pedigrees, because this larger deletion would cover both of the above regions. Additionally, the non-lethal pedigree BG contains two separate deletions not immediately adjacent to the donor site, minimally defined by FISH, suggesting that these regions do not harbor essential sequences. Two cases of insertion translocations have been identified as demonstrated in pedigree AG (Figure 6C) and a previously described translocation involving just the transposon concatemer [8]. Finally, pedigree BC harbors both inversions and deletions, defined by FISH (Figure 6D) and ROMA (Figure 7B). In summary, out of nine examined cases of transposon mobilization on Chromosome 11, every pedigree demonstrated some form of genomic rearrangement, spanning hundreds of thousands to tens of millions of base pairs.

RT-PCR and standard PCR genotyping.

RNA was extracted and RT-PCR performed as described [8]. Transposase transgene genotyping was done using standard techniques on tail biopsy DNA. Individual insertion genotyping was done as previously described using three-primer PCR [39]. Primer sequences for all PCR reactions are available upon request.

Immunohistochemistry.

Sections were cut from frozen muscle on a Tissue Tek II microtome/cryostat (Miles Scientific, Naperville, Illinois), fixed, and stored at −20 °C until use. Slides were air dried followed by incubation in permeabilizing/blocking solution (P/BS) (0.12% BSA, 0.12% nonfat dry milk, 0.1% Triton X-100 in phosphate-buffered saline [PBS]) plus 5% normal goat serum for 1 h at 4 °C and then rinsed three times in PBS. SC-71 (used as an undiluted hybridoma supernatant) reacts with MyHC-IIa (Myh2), NCL-MHCs (Novocastra Laboratories Newcastle-upon-Tyne, United Kingdom, 1:50 dilution) reacts with MyHC-I (Myh7), and Anti-Laminin (Sigma-Aldrich, St. Louis, Missouri, United States, 1:200 dilution) aided in muscle fiber identification. Primary antibody incubations were carried out at room temperature for 2 h, washed twice with PBS and incubated twice for 5 min in P/BS. Secondary antibody incubations were performed in P/BS at room temperature for 2 h using Goat-anti-mouse FITC- or TR-conjugated IgG (Jackson ImmunoResearch, West Grove, Pennsylvania, United States, 1:100 dilution) for Anti-MyHC detection and FITC-conjugated goat-anti-rabbit (Jackson ImmunoResearch, 1:100 dilution) for Anti-Laminin detection. Sections were washed 3 times with PBS, incubated twice for 5 min in PBS, and mounted in glycerol-gelatin (Sigma-Aldrich).

Fluorescence in situ hybridization.

129Sv library BAC clones obtained complementary of The Wellcome Trust Sanger Institute (Cambridge, United Kingdom) and the pT2/GT3/tTA transposon-harboring plasmid [8], were labeled with biotin or digoxigenin, respectively, by nick translation as we have previously described [1]. Probes were detected using rhodamine-conjugated strepavidin or fluorescein-conjugated anti-digoxigenin antibody, respectively (Rainbow Scientific, Windsor, Connecticut, United States), as also described by Carlson et al [1].

The authors would like to thank all the members of The Arnold and Mabel Beckman Center for Transposon Research for their commitment to valuable discussions and suggestions. We thank Drs. Adam Dupuy, Nancy Jenkins, and Neal Copeland for providing the RosaSB11 strain of mice, Drs. Robert Lucito, Lakshmi Muthuswamy, and Michael Wigler, for developing the mouse ROMA chip and bioinformatic tools for its analysis, and Sandra Horn, Cesilie Granum, and the Mouse Genetics Laboratory for their expertise in producing transgenic mice.