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

使用 RLGS（限制性地标基因组扫描）克隆与神经元再生和可塑性相关的新基因

• 批准号: 08671605
• 负责人: YAMAKI Tarumi
• 金额: $ 1.41万
• 依托单位: Kyoto Prefectural University of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671605/
• 关键词: Diffuse axonal injury; RLGS; Plasticity; Diffuse brain injury; Cloning; 実験的びまん性脳損傷; 神経再生; 神経可塑性; RLGS法

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.

我们已经开发了我们的原始设备（Yamaki等人，J.Neurotrauma，1994）以产生弥漫性脑损伤的大鼠模型，其显示出与人弥漫性轴索损伤相似的病理学发现。我们一直在分析这些模型大鼠的分子生物学特征和记忆和行为障碍。本研究的目的是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。使用Hayashizaki等（Electrophoresis 14，1993）开发的RLGS（限制性标志基因组扫描）方法，并稍作修改。近年来，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是通过比较样品和对照之间的甲基化差异来发现基因组DNA上转录激活或失活部分的方法。在最初的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。而我们发展了非放射性同位素方法，在该方法中，用地高辛-ddUTP标记DNA的切割末端，并使用碱性磷酸酶缀合的抗地高辛抗体的化学发光系统检测标记。虽然我们发现在这些标记和检测过程中存在困难，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，我们继续在我们的大鼠模型中筛选激活的基因表达。使用损伤后18天和25天的大鼠大脑内嗅皮层和海马，因为预计损伤神经元中的突起再生在损伤后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天找到一些激活基因的片段，现在我们正急于确定这些克隆的序列。