Developmental Study on a Real-time Measurement of Gene Transcriptional Activity by Means of lon-channel Reporter Genes.

基因转录活性的调节已通过连接到基因编码区5'上游的各种报告基因进行分析。报道基因一般为β-半乳糖苷酶、过氧化氢酶、荧光素酶基因，通过匀浆酶活性检测或固定后组织化学方法检测。为了分析活细胞中的转录活性，离子通道基因是非常合适的，因为离子通道的活性可以在活状态下电生理地测量，并且有时可以通过适当的荧光指示剂通过细胞内离子浓度的变化来光学地检测。本课题的目的是探讨离子通道基因作为报告基因在海鞘双细胞模型神经诱导系统中应用的可能性。获得的结果是：（1）为了利用海鞘钠通道基因作为报告基因，我们从海鞘幼虫cDNA文库中克隆了TuNa Ⅰ基因，并测定了其全序列。对TTX不敏感可能是由于SS 2区的点突变所致。(2)连接到RSV启动子的TuNa I的cDNA克隆给我们可变的表达效率，我们从cDNA合成TuNa I mRNA注射到海鞘神经卵裂球中以阐明可变性的原因。(3)将胸苷激酶mRNA编码区5 '端的一部分与TuNa Ⅰ mRNA编码区的头部连接，可获得较高的表达效率，并认为TuNa Ⅰ至少在海鞘胚胎中可用作报告基因。(4)为了区分TuNa I的外源性表达和内源性表达，我们尝试通过SS 2区域的定点突变将TTx不敏感通道改变为TTX敏感通道。

Regulation of transcriptional activities of a gene has been analyzed by means of various reporter genes linked to 5' upper stream of the coding region of the gene. The reporter genes generally used are beta-Gal, CAT,Luciferase genes, which are detected by the enzymatic activities in homogenates or by histochemical methods after fixation. In order to analyze transcriptional activities in living cells ion channel genes are extremely suitable because activities of ion channels can be measured electrophysiologically in living state and sometimes may be detected optically through changes in intracellular ion concentrations by appropriate fluorescent indicators. Aim in the present project is to test the possibility of using ion channel genes as reporter genes in the model neural induction system of the ascidian two-cell pair. Obtained results are : (1) In order to use an ascidian Na channel gene as a reporter we cloned TuNa I from larval cDNA library and whole nucleotide sequence was determined. The insensitivity to TTX was suggested to be due to a point mutation in SS2 region. (2) The cDNA clone of TuNa I linked to RSV promotor gave us variable expression efficiency and we synthesized TuNa I mRNA from cDNA to inject ascidian neural blastomeres to clarify causes of the variability. (3) We found high efficiency of expression, when a part of 5'end of the coding region of thymidine kinase mRNA was linked to the head of the coding region of TuNa I mRNA,and concluded that TuNa I was useful for a reporter gene at least in ascidian embryos. (4) In order to discriminate exogenous expression of TuNa I from the endogenous one we have tried to altemate the TTx insensitive channels to the TTX sensitive ones by site-directed mutagenesis in the region of SS2.