| Summary: | 碩士 === 國立陽明大學 === 神經科學研究所 === 91 === Abstract
Neural stem cells are involved in neural development and are capable of generating a variety of neural cells. Previously, we used a subtractive hybridization assay, in which cDNA of embryonic day 10.5 (E10.5) rat neural tube was subtracted from cDNA of postnatal day 1 (P1) rat brain, to identify genes preferentially expressed in early neural tube. Neural stem cell marker 1 (NSC1) gene was identified in this assay and was hypothesized as a membrane receptor.
In this project, I intended to detect the expression pattern of NSC1. First, I used in situ hybridization in different stages of rat embryos to detect NSC1 expression pattern. In the E9 stage, NSC1 was mainly expressed in neuroepithelium. Later in the E10 embryo, NSC1 gene was expressed in neuroepithelium, developing neural tube and somites. In the E10.5 stage, expression of the NSC1 gene was also strong in neural tube. However, the expression level in somites was reduced. In the E11.5 stage, the expression level in neural tube was down-regulated and the signals in somites were hardly detected. In the E12.5 stage, NSC1 expression was barely detected in the neural tube. Second, I generated monoclonal antibodies (mAbs) against NSC1. I had tested hundreds of clones for their specificity by western blotting and immunocytochemistry. Two clones, 6B4 against NSC1 extra-cellular domain and 1D5 against NSC1 intra-cellular domain, were selected and purified. It was shown that 6B4 and 1D5 were specifically against NSC1 in western blotting and immunocytochemistry. Results from cell fluorescence staining of HEK293T cells overexpressing NSC1 showed that staining of NSC1 was on the cell membrane, as demonstrated in the confocal microscopy and the live staining. Immunohitochemistry staining of different stages of embryos showed that NSC1 was expressed in neuroepithelium and neural tube in the early stage embryo. Expression of NSC1 protein was reduced in the later stage embryo. From the data of in situ hybridization and immunohitochemistry, expression of NSC1 was in a developmentally regulated pattern.
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