1996 Fiscal Year Final Research Report Summary
A gene trap approach to identify a new gene involved in central nervous system development
| Project/Area Number |
06454698
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Niigata University |
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Principal Investigator |
KUWANO Ryozo Niigata University Research Laboratory for Molecular genetics Associate Professor, 遺伝子実験施設, 助教授 (20111734)
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| Project Period (FY) |
1994 – 1996
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| Keywords | gene / gene trap / nervous system development / ES cells / neomycin resistant / chimeric mice / embryonic leathal / immunohistochemistry |
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| Research Abstract |
A gene trap approach is used to identify a new gene involved in development of central nervous system. A trap vector was constructed with a splicing acceptor of mouse E-cadherin gene, neomycin ribosyl transferase (neo), beta-galacosidase (lacZ) and IRES derived from encephalomyocarditis virus gene. The gene trap vector were introduced into mouse embryonic stem (ES) cells by electroporation. ES cells were selected with G418 and allowed to differentiate in suspension culture system and immunohistochemically examined with antibodies against brain specific proteins (nestine, neurofilament, MAP2 and glial fibrillary acidic protein). Of 13 G418 resistant cell lines 3 cell lines (GT3-8,11 and 12) were immunoreactive and simultaneously manifested lacZ gene expression.
Chimeric mice were generated by injection of the 13 ES cell lines into wild type C57BL/6 blastulas and an expression pattern of the trap gene was analyzed at 14.5 dpc embryos of chimeric mice. Among them lacZ expression was detected in the central nervous system of GT3-8,11, and 12. The GT3-11 and 12, but not GT3-8, were transmitted into the germ line. The trapped genes were isolated from ES cells by plasmid rescue and the cDNA fused with the gene trap vector were obtained by 5'RACE.Novel gene were trapped in both GT3-11 and 12 from these partial DNA sequences and data base search. Cloning of the full length cDNAs and the trapped gene and their structural analyzes are now in progress.
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[Publications] S.Kawahire, M.Takeuchi, T.Gohshi, S.Sasagawa, M.Shimada, M.Takahashi, T.K.Abe, T.Ueda, R.Kuwano, A.Hikawa, T.Ichimura, S.Omata and T.Horigome: "cDNA cloning of nuclear localization signal binding protein NFP60, a rat homologue of lamin B receptor, and identification of binding sites ofhuman lamin B receptor for" J.Biochem.(in press).
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