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Influence of congenital hydrocephalus on embryonic brain development and its intrauterine therapy

先天性脑积水对胚胎脑发育的影响及其宫内治疗

基本信息

批准号：
13470289
负责人：
HAYASHI Nakamasa
金额：
$ 8.83万
依托单位：
Toyama Medical and Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470289/
关键词：
TGF beta 1 Cre recombinase EGFP GFAP promoter Western blot RT-PCR transgenic mouse Hydrocephalus マイクロインジェクション Creリコスビナーゼ lox P TGF-beta 1 hydrocephalus mouse TGF-beta1 transgenic mouse embryo

项目摘要

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.

1.我们制造了转基因3.以EGFP和TGF β 1为探针，进行脑RNA的北方印迹分析，结果表明：（1）用GFP和TGF β 1为探针，将GFP和TGF β 1基因分别导入小鼠卵母细胞原核内，获得了4个转基因小鼠品系。3个品系的转基因小鼠均表达EGFP mRNA，但TGF β 1 mRNA均不表达。4.将GFAP-EGFP-TGF转基因小鼠与表达Cre重组酶的转基因小鼠交配。通过基因组PCR和Southern blot分析，在第二代小鼠中，EGFP-SV 40基因的填充序列被切除，因此我们将该小鼠命名为“GFAP-TGF转基因小鼠”。5.为了确定切除后的GFAP-TGF转基因小鼠中TGF β 1基因是否被激活，我们以脑RNA为模板进行了RT-PCR分析。RT-PCR检测结果表明，TGF β 1基因在GFAP-TGF转基因小鼠脑中有过表达，为了确定TGF β 1蛋白在GFAP-TGF转基因小鼠脑中是否有表达，我们对脑组织提取物进行了Western blot分析。Western blot分析显示，GFAP-TGF转基因小鼠脑内TGF β 1蛋白表达增加。7.本研究利用Cre/loxP系统建立了稳定表达人GFAP启动子控制下突变型TGF β 1蛋白的转基因小鼠。通过将该转基因小鼠作为纯合子或与Cre转基因小鼠进一步交配，我们将能够建立类似于人类先天性脑积水的高贵小鼠模型。