Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers

Abstract

Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes.

Figure 1. Design of a Forward-Genetic Screen

(A) The T2/GT3/tTA gene-trap tTA transposon was designed with splice acceptors (SA) in both orientations and the bidirectional SV40 polyadenylation signal (pA) to truncate expression of an endogenous gene after insertion into an intron. LoxP recombination sites (gray arrowheads) flank the mutagenic core of the transposon to potentially rescue a transposon-induced mutation. (B) Southern blot and PCR analysis (+/–, top) was used to identify G1 animals that inherited the concatemer, but not the transposase transgene (asterisk). (C) Insertions (red rectangle) genetically linked to the concatemer donor site (black rectangles) on Chromosome 11 (—) are homozygosed in a three-generation breeding scheme using the Inv(11)8Brd^Trp53–Wnt3 strain balancer chromosome (↔) with its engineered Wnt3 mutation and visible Agouti marker conferring a yellowish color to the ears and tail. G1 animals were crossed to mice that carry a balanced Rex (curly coat) mutation (gray outline). Animals inheriting two copies of the balancer die in utero.

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.

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).

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.

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).

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.

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 8. De Novo Rearrangements in Somatic Cells of a GT3A; RosaSB11 Mouse

Metaphase and interphase FISH images of normal (A, C, E) and abnormal (B, D, F) splenic lymphocytes from a doubly transgenic mouse are shown using the same probes as Figure 6. Evidence for deletion (white arrows) and translocated Chromosome 11 sequences (yellow arrows) were evident for these three probes. These data, including other probes, are further summarized in Table S2.