Analysis of transgenerational effects on DNA copy number aberrations in male mice exposed to continuous 0.05mGy/day gamma-rays for 400 days (Secondary screening for 0.05mGyJ familly).

Transgenerational effects of continuous low dose-rate (LDR) gamma-ray irradiation have not been well studied. Recent advances in DNA technology enabled us to examine a whole genome at molecular level. Here we adopted one of these techniques called oligo-microarray comparative genome hybridization (CGH) and studied trans-generational effects on DNA copy number aberrations (CNAs). C57BL/6JNrs male mice were exposed to LDR gamma-rays (0.05 mGy/day) for 400 days (total dose: 20 mGy) from 8 weeks of age. Progeny from 0.05 mGy/day irradiated mice had significantly lower frequencies of genomic aberrations than the progeny of non-irradiated mice.This study investigated the De novo mutation by comparing the parents and children using 15 LDR gamma-rays (0.05 mGy/day) irradiated male family and 25 non irradiated male family. This is a secondary screening. This study was performed under contract with the Aomori Prefectural Government, Japan. Two-condition experiment, C57BL/6JNrs tail tissue non-irradiated male (0.05 mGyJM), female (0.05 mGyJF) and their progenies (0.05 mGyJ1 to 0.05 mGyJ6) vs. reference male mouse. We irradiated and non-irradiated male mice (C57BL/6JNrs) for 400 days and cross to non- irradiated female mice (C57BL/6JNrs) and got F1 mice. After they dead the DNA were prepared from their tails. At first, primary screening using １M oligo-microarray was performed. The array was mounted autosomal fragment at intervals of an average about 2kb. Two-condition experiment, mice tail tissue irradiated or non-irradiated male, female and their progenies vs. reference male mouse. This reference male mouse was from the same colony used in this experiment. Twice by changing the labeling color Cy3 and Cy5, performed CGH and extracting probes showed either aberration. Identification the chromosomal location of the probe. Probes adjacent to the probe with aberration were set at high density. The candidate probes in one family was designed in a single 4x 244k or 8x 60k oligo-microarray and performed secondary screening. Extracting the flanking probes showed either aberration when labeled with Cy3 and Cy5 and identification the chromosomal location of the probe. Finally, we performed TaqMan® Copy Number Assays to verify the mutations.

持续低剂量率（low dose-rate, LDR）γ射线辐照的跨代效应尚未得到充分研究。近年来DNA技术的进步使我们得以在分子层面检测全基因组。本研究采用了其中一项名为寡核苷酸微阵列比较基因组杂交（oligo-microarray comparative genome hybridization, CGH）的技术，探究了辐照对DNA拷贝数变异（copy number aberrations, CNAs）的跨代效应。选取8周龄的C57BL/6JNrs雄性小鼠，使其接受每日0.05 mGy的持续低剂量率γ射线辐照，持续400天，总辐照剂量达20 mGy。接受辐照的雄性小鼠的子代，其基因组异常频率显著低于未辐照雄性小鼠的子代。本研究通过对比15个接受0.05 mGy/天持续低剂量率γ射线辐照的雄性家系与25个未辐照雄性家系的亲代与子代，对新发突变（de novo mutation）进行了分析。本研究属于二次筛选。本研究依托与日本青森县政府签订的合作协议开展。实验采用双条件对照设计：以C57BL/6JNrs小鼠尾部组织为样本，分别选取未辐照雄性（0.05 mGyJM）、未辐照雌性（0.05 mGyJF）及其子代（0.05 mGyJ1至0.05 mGyJ6），与参照雄性小鼠进行对照。我们对C57BL/6JNrs雄性小鼠进行为期400天的辐照与未辐照处理，随后将其与未辐照的C57BL/6JNrs雌性小鼠交配，获得F1代小鼠。待小鼠死亡后，提取其尾部组织的DNA。首先，采用1M寡核苷酸微阵列开展初步筛选。该微阵列以平均约2 kb的间隔排布常染色体探针片段。本次实验采用双条件对照设计：以辐照或未辐照的雌雄小鼠及其子代的尾部组织为样本，与来自本实验同一饲养种群的参照雄性小鼠进行对照。通过交替更换标记染料Cy3与Cy5，完成两轮比较基因组杂交实验，筛选出显示异常信号的探针。对各探针的染色体定位进行鉴定。针对存在异常信号的探针，将其邻近探针设置为高密度排布。单个家系的候选探针被设计于单张4×244k或8×60k寡核苷酸微阵列上，以此开展二次筛选。使用Cy3与Cy5进行标记后，筛选出显示异常信号的侧翼探针，并再次鉴定其染色体定位。最终，采用TaqMan®拷贝数检测试剂盒（TaqMan® Copy Number Assays）对突变进行验证。