Abstract
Several methods are known to isolate radiosensitive gene such as identification of responsible gene mutations in radiosensitive genetic disorder, homologue analysis from model organism, biochemical analysis of intermolecular interaction, differential display, and subtraction method. However, these methods have peculiarity in isolation, so we have designed more efficient methods to isolate radiosensitive genes.
We call it RAZE (Radiosensitivity-related gene cloning using Zeosin vector) method, which enables to efficiently obtain DNA damage resistant cell strains. The hallmark of this method is the random insertion of zeosin vector, which leads to establish genetically modified cell strains. We first transfected our original zeosin vector into highly radiosensitive MOLT-4 cells by electropolation. The vector we constructed is specialized for inverse PCR (IPCR), which are used to specify vector's insertion sites in genomic DNA. It consists of zeosin resistant gene with multiple restriction sites in both ends. We then 10 Gy-irradiated those transfectants and evaluated its viability. We selected clones that are more than 2-fold viability to parental cell line. Since we proved that the amount of inserted zeosin-resistant gene has no correlation with its radioresistance, we concluded that acquired radioresistance is due to its DNA modification by zeosin vector. Thus, we identified zeosin vector's insertion sites in these radioresistant clones using IPCR. Multiple restriction sites enabled to create various DNA fragments, which were advantage to IPCR. We successfully isolated the disrupted genes of interest. Our efforts have been directed toward a functional analysis of the candidate genes.
