| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
The screen and analysis of behavioral mutants has been a fruitful approach in identifying genes involved in function and/or development of nervous system. However, genes whose disruption lead to the lethality, or genes which are present redundantly in genome could not be identified by the conventional screen for viable mutants. In this study, I have attempted to identify novel neuronal genes by establishing an ectopic expression system in a worm, C.elegans. For this purpose, I have constructed C.elegans cDNA libraries which are expressed under a neuronal or an ubiquitous promoter/enhancer, have introduced the libraries into C.elegans animals, and examined the phenotype of the transgenic worms.
Two expression vectors with an EcoRI-NotI directional cloning site have been constructed using genomic fragments responsible for the tissue specific expression. To drive expression in neuronal tissues, H20, a genomic fragment which was isolated by promoter trapping by Dr.Takeshi Ishihara at NIG in
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Japan, was used. For expression in ubiquitous tissues, EF1a, which is a 3kb genomic fragment upstream to the C.elegans elongation factor 1a gene isolated by Dr.Makoto Koga at Kyushu University in Japan, was used. cDNAs synthesized from poly (A) + RNA isolated from mixed-stage worms were inserted into the directional cloning site of the vectors, and libraries consisting of 6 x 10^5 individual clones were constructed. Plasmid DNA extracted from cDNA pools each consisting of around 250 clones were injected together with a transformation marker, pRF4, into the distal arms of adult hermaphrodites. The recipient ST2 is a transgenic strain whose entire nervous system was visualized by the expression of GFP.For each pool, more than 10 F2 or F3 progenies of independently established transformant lines were examined for their overall morphology and movement under a dissection microscope, and for the morphology of the nervous system under a fluorescent microscope.
Three thousands clones have been examined for each libraries. No clone from the library with H20 caused an abnormality at F2 progenies. A F1 transformant with pool #9 from the library with EF1a gave rise to 20 F2 worms, of which 16 were abnormal ; they had protrusion at the presumptive vulval region at L3 stages, and then later limped and appeared dehydrated. They died without laying eggs. I have injected the same pool two more times, but no F1 or F2 transformants showed similar defects. Less
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