| Project/Area Number |
18500327
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory animal science
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| Research Institution | Osaka University |
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Principal Investigator |
KOKUBU Chikara Osaka University, Graduate School of Medicine, Assistant Professor (70379238)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Developmental engineering / Chromosomal engineering / cis-regulatory element / Enhancer / Morphogenesis / Transposon / Mouse / Heterochromatin |
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| Research Abstract |
Large cisregulatory landscapes are a remarkable feature of vertebrate genomes, particularly seen at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying such a large genomic region of interest, ranging from a few kb to a few Mb, are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping property of the Sleeping Beauty (SB) transposon in the mouse genome. We devised the transposon-based Local Hopping Enhancer Detector (LHED) vector carrying an enhancer-detection LacZ reporter and loxP cassette, which allows monitoring of transcription-enhancing activities in the native genomic context and Cre-mediated deletion/inversion rearrangement between the transposon donor and acceptor sites.
As a proof of principle, we generated embryonic stem (ES) cells with a targeted integration of the LHED vector into the developmentally and evolutionally critical Paxl gene locus, followed by efficient local transposition, nested deletion formation, and rapid derivation of embryos by tetraploid complementation. Whole-mount reporter expression analysis of the engineered embryos showed different expression patterns corresponding to each insertion/deletion configuration and markedly facilitated long-range cis-regulatory element mapping in this genomic neighborhood, demonstrating the great potential of this experimental platform for functional exploration of very focused areas in the mouse genome (manuscript in revision).
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