Abstract
Recent consolidation of the whole-genome sequence with genome-wide transcriptome profiling revealed the existence of functional units within the genome in specific chromosomal regions, as seen in the coordinated expression of gene clusters and colocalization of functionally related genes. An efficient region-specific mutagenesis screen would greatly facilitate research in addressing the importance of these clusters. Here we use the 'local hopping' phenomenon of a DNA-type transposon, Sleeping Beauty (SB), for region-specific saturation mutagenesis. A transgenic mouse containing both transposon (acts as a mutagen) and transposase (recognizes and mobilizes the transposon) was bred for germ-cell transposition events, allowing us to generate many mutant mice. All genes within a 4-Mb region of the original donor site were mutated by SB, indicating the potential of this system for functional genomic studies within a specific chromosomal region.
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Acknowledgements
We thank G. Kondoh, M. Kouno, E.S. Saito and R. Ikeda for helpful advice and excellent technical assistance. We also thank S. Makino and Y. Odan for administrative assistance in preparing this manuscript. This work was supported by grants from the New Energy and Industrial Technology Development Organization of Japan; Uehara Memorial Foundation; Preventure Program, Japan Science and Technology Agency; RIKEN, The Institute of Physical and Chemical Research; and a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Supplementary information
Supplementary Fig. 1
Examples of two phenotypic screens generated by SB transposon system. (PDF 72 kb)
Supplementary Fig. 2
Determining the exact location of IR3 donor site on chromosome 12. (PDF 85 kb)
Supplementary Fig. 3
Gene trap and region-specific saturation mutagenesis using the SB transposon system for IF2 donor mouse. (PDF 214 kb)
Supplementary Fig. 4
Detection of new transcripts using the SB transposon system. (PDF 90 kb)
Supplementary Fig. 5
Screen shot of the UCSC Mouse Genome Browser Version mm4 with our SB database overview of transposon insertion sites for IR3 and IF2 donor mice. (PDF 136 kb)
Supplementary Table 1
List of primers used in the nested-PCR. (PDF 54 kb)
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Keng, V., Yae, K., Hayakawa, T. et al. Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods 2, 763–769 (2005). https://doi.org/10.1038/nmeth795
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DOI: https://doi.org/10.1038/nmeth795