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Molecular biological studies of Ten-m2 gene function for the olfactory sensory neuron projections

嗅觉感觉神经元投射的 Ten-m2 基因功能的分子生物学研究

基本信息

批准号：
12470356
负责人：
OOHASHI Toshitaka
金额：
$ 9.41万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

To investigate the function of ten-m2/Neurestin gene for the olfactory sensory neuron projection, we have done the following experiments and got some data.Ten-m2 specific antibodies were raised using the C-terminal unique amino acid sequence among the ten-m family. The specific staining was confirmed in the mouse section and confirmed its embryonic expression in combination with in situ hybridization.Detection of Ten-m2 ligand: The extracellular domain of Ten-m2 protein was expressed in HEK293 cell culture system. We could detect specific protein band in the SDS-PAGE which bound to the ecto-domain. We are now identifying the protein.Chromosomal mapping of human ten-m2 gene. The ten-m2, 3, and 4 genes were mapped to the chromosome 5, 4and 11, respectively. There was no genetic disorder reported to the locus.Phenotypic analysis of the ten-m2 knockout mouse: By introducing a neomycin expression cassette in to the transmembrane exon of type II transmembrane protein, ten-m2, ten-m2 null mice were generated. There was no obvious phenotype so far. Ten-m2 null mice were viable and fertile, had a normal life span. There could be some compensation by another ten-m family members. The olfactory neuronal axon is now stained with MBP, marker of myelin and observed carefully.Another approach to look for the ligand for ten-m2: The ten-m protein seems to be quite unique for its protein structure. Structural analysis will become a very important information to search the ligands. Ultrastructural analysis of recombinants revealed the dimerfomation through the disufide-bond. The dimer could be formed between the different members of ten-m family in our reconstitution experiments. Since CSPGs are known to act for the axon guidance, and the versican is expressed in the olfactory bulb, we tried to clone some related genes. The Brain link protein is cloned and the expression pattern was analyzed. The data for the novel link protein is published (see references).

为了研究ten-m2/Neurestin基因在嗅觉神经元投射中的作用，我们进行了以下实验并获得了一定的数据：利用ten-m家族C端的独特氨基酸序列，制备了Ten-m2特异性抗体。在小鼠切片中进行特异性染色，并结合原位杂交证实其在胚胎中的表达。Ten-m2配体的检测：Ten-m2蛋白胞外区在HEK 293细胞培养系统中得到表达。在SDS-PAGE上可检测到与胞外区结合的特异性蛋白条带。我们现在正在鉴定蛋白质。人类ten-m2基因的染色体定位。10个m2、3和4基因分别定位在第5、4和11染色体上。ten-m2基因敲除小鼠的表型分析：通过将新霉素表达盒引入II型跨膜蛋白的跨膜外显子中，产生ten-m2、ten-m2缺失小鼠。到目前为止还没有明显的表型。10-m2基因敲除小鼠存活并具有生育能力，具有正常的寿命。另外1000万家庭成员可能会给予一些补偿。用髓鞘标记物MBP对嗅神经元轴突进行染色并仔细观察。另一种寻找ten-m 2配体的方法：ten-m蛋白的结构似乎很独特。结构分析将成为寻找配体的重要信息。重组子的超微结构分析显示二聚体通过二硫键形成。在我们的重组实验中，ten-m家族的不同成员之间可以形成二聚体。由于已知CSPGs起轴突导向作用，并且多功能蛋白聚糖在嗅球中表达，我们试图克隆一些相关基因。克隆了Brain link蛋白并分析了其表达模式。新连接蛋白的数据已发表（参见参考文献）。