| Co-Investigator(Kenkyū-buntansha) |
KURIMOTO Masanori Toyama medical and Pharmaceutical University, University Hospital, Assistant Professor, 附属病院, 講師 (10161770)
ENDO Shunro Toyama medical and Pharmaceutical University, Faculty of Medicine, Professor, 医学部, 教授 (70125269)
HIRASHIMA Yutaka Toyama medical and Pharmaceutical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (30135016)
KATO Ichiro Toyama medical and Pharmaceutical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (50250741)
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| Research Abstract |
1.We made the transgene(GFAP-EGFP-TGF) that carry human GFAR promoter, loxP,EGFP DNA,SV40 DNA,loxP,mutantTGF beta1 cDNA, SV40 DNA in this sequence.
2.We injected the transgene into mouse egg pronuclei by DNA microinjection method and established 4 lines of transgenic mice that transmit the transgene through the germ-line.
3.In Northern blot analysis of brain RNA using EGFP and TGF bata1 as DNA probes, EGFP mRNA but not TGF beta1 mRNA was strongly expressed in the 3 lines of transgenic mice.
4.Next, we mated GFAP-EGFP-TGF transgenic mice with Cre recombinase-expressing transgenic mice. In the next generation, excision of stuffering EGFP-SV40 sequence was observed by genome PCR and Southern blot analyses and so we designated this mouse 'GFAP-TGF transgenic mice'.
5.To determine whether the TGF beta1 gene is activated in the post-excision GFAP-TGF transgenic mice, we performed RT-PCR analysis using brain RNAs as templates. RT-PCR analysis indicated that the TGF beta1 gene is overexpressed in the GFAP-TGF transgenic mice.
To determine whether the TGF beta1 protein is expressed in the brains of GFAP-TGF transgenic mice, we performed Western blot analysis using brain extracts. Western blot analysis indicated that the TGF beta1 protein is increased in the brains of GFAP-TGF transgenic mice.
7.In the present study, by using the Cre/loxP system, we established the stable transgenic mice that overexpress the mutant TGF beta1 protein under the control of human GFAP promoter. By making this transgenic mice as homozygotes or further mating with Cre-transgenic mice, we will be able to establish noble mouse model that resemble the human congenital hydrocephalus.
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