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

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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.