Cloning of new gene related to neuronal regeneration and plasticity using RLGS (Restriction Landmark Genomic Scanning)

我们开发了我们的原始装置(Yamaki等人，J.Neurotragation，1994)来制作弥漫性脑损伤的大鼠模型，其病理结果与人类弥漫性轴索损伤相似。我们一直在分析这些模型大鼠的分子生物学特征以及记忆和行为障碍。本研究计划在我们的模型中寻找与再生过程和可塑性变化密切相关的新基因。限制地标基因组扫描(RLGS)方法是由Hayashizaki等人发展起来的。(电泳法14,1993)，略有修改后使用。近年来，人们普遍注意到基因组DNA的甲基化和去甲基化在基因表达调控中起着重要作用。RLGS是一种通过比较样本和对照之间的甲基化差异来寻找基因组DNA上转录激活或失活部分的方法。在原始的RLGS方法中，基因组DNA用甲基化敏感的限制性内切酶切割，然后用放射性同位素标记切割的末端。然而，我们发展了一种非放射性同位素方法，即用地高氧素-ddUTP标记DNA的切端，并用抗地高氧素抗体与碱性磷酸酶结合的化学发光体系进行检测。虽然我们在这些标记和检测过程中发现了困难，但我们可以设法优化反应条件，并在高灵敏度X射线胶片上获得最终的斑点图案。接下来，我们继续筛选我们的大鼠模型中激活的基因表达。使用大鼠脑损伤后18天和25天的内皮层和海马区，因为损伤神经元突起的再生在损伤后18天最显著，神经元连接的重建在损伤后25天最活跃。我们可以在损伤后18天和25天发现几个激活基因片段，现在我们正在匆忙确定这些克隆的序列。

We had developed our original device (Yamaki et al., J.Neurotrauma, 1994) to produce rat model of diffuse brain injury, which showed similar pathological findings to human diffuse axonal injury. We have been analyzing molecular-biological features and memory and behavioral impairments of these model rats. The present study was planed to find new genes deeply related to regeneration processes and plastic changes in our models. RLGS (Restriction Landmark Genomic Scanning) method, which was developed by Hayashizaki et al. (Electrophoresis 14,1993), was used with slight modifications. Recently, it is generally noted that methylation and demethylation on genomic DNA play an important role in regulating gene expression. RLGS is the method to find transcriptionally activated or inactivated portions on genomic DNA by comparing differences of methylation between samples and the controls. In the original method of RLGS,genomic DNA was cut with methylation-sensitive restriction enzyme followed by labeling the cut end with radioisotope. However, we developed non-radioisotope method, in which cut end of DNA was labeled with digoxygenin-ddUTP,and the labeling was detected with chemiluminescence system using anti-digoxygenin antibody conjugated with alkaline phosphatase. Although we found difficulties in these labeling and detection process, we could manage to optimize the reaction conditions and get final spot patterns on high-sensitivity X-ray films. Next, we proceeded to screen activated gene expressions in our rat models.Entorhinal cortex and hippocampus of rat brains 18 days and 25 days after injury were used, because regeneration of processes in injured neurons was expected to be most prominent in 18 days and reconstruction of neuronal connections was most activated in 25 days after injury. We could find a couple of fragments of activated genes in both 18 days and 25 days after injury, and now we are in a hurry to determine sequences of those clones.