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.
Highlights
The Sleeping Beauty (SB) transposable element is an effective tool for generating mutations in the germline [1,2,3,4,5] and somatic cells [6,7] of mice
Generation of Mice for a Forward-Genetic Screen The protocol for SB-mediated germline mutagenesis has been described by multiple groups [2,3,13]
Founder line 6660 was previously mapped to chromosome 11, band 11C, by fluorescence in situ hybridization (FISH), and harbors a concatemer of approximately 30 copies of the transposon vector [8]. We renamed this line the ‘‘GT3A’’ line and used two different strains of transgenic mice to mobilize these transposons from their Chromosome-11 donor site in the germline of mice
Summary
The Sleeping Beauty (SB) transposable element is an effective tool for generating mutations in the germline [1,2,3,4,5] and somatic cells [6,7] of mice. Previous studies indicate that the advantages of this cut-and-paste transposon system are four fold. Insertional mutations in genes can be identified and tracked through generations using simple PCR-based techniques because the transposon vector serves as a molecular tag. A recent study achieved germline saturation mutagenesis by mobilizing chromosomally resident transposons in a region of mouse Chromosome 12 [5]. A fourth advantage is that the transposon vector can be designed to include functional elements that report expression patterns of mutated genes [4,8]. Previous strategies for the use of SB for germline mutagenesis have used reverse-genetic approaches, whereby potential mutations have been selected based on sequence information [1,8] or expression analysis [4,5]. The goal of the study presented here, rather, was to test whether the SB transposon system could be used as a mutagen for forward-genetic studies in the mouse germline
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