Analysis of transgenerational effects on DNA copy number aberrations in male mice exposed to continuous 0.05mGy/day gamma-rays for 400 days (Primary screening for 0.05mGyB 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 primary 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 mGyBM), female (0.05 mGyBF) and their progenies (0.05 mGyA1 to 0.05 mGyB8) 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（LDR）γ射线照射的跨代效应尚未得到充分研究。近年来DNA技术的进步使得我们能够在分子层面实现全基因组检测。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（CGH）的技术，探究了跨代DNA拷贝数变异（CNAs）的效应。
将8周龄的C57BL/6JNrs雄性小鼠以0.05 mGy/天的剂量接受为期400天的LDRγ射线照射（总剂量：20 mGy）。经照射雄性小鼠的子代，其基因组变异频率显著低于未照射对照组小鼠的子代。
本研究通过对比15个LDRγ射线照射（0.05 mGy/天）的雄性家系与25个未照射雄性家系的亲子样本，对新发突变进行探究，该实验属于初步筛选阶段。本研究与日本青森县政府合作开展。
本实验采用双组对照设计：以C57BL/6JNrs尾组织来源的未照射雄性小鼠（0.05 mGyBM）、雌性小鼠（0.05 mGyBF）及其子代（0.05 mGyA1至0.05 mGyB8）为实验组，与同批次参照雄性小鼠进行对照。
我们对雄性C57BL/6JNrs小鼠进行为期400天的照射与未照射处理，随后与未照射的雌性C57BL/6JNrs小鼠交配，获得F1代小鼠。待F1代小鼠死亡后，提取其尾部组织的基因组DNA。
首先采用1M寡核苷酸微阵列开展初步筛选。该芯片以平均约2kb的间隔排布常染色体片段。本次比较基因组杂交实验采用双组对照设计：以照射或未照射的雌雄小鼠及其子代的尾组织为样本，与同批次参照雄性小鼠进行对照。该参照雄性小鼠来自本实验所用的同一繁育种群。
通过更换荧光标记染料Cy3与Cy5，分两轮完成比较基因组杂交，筛选出存在变异的探针，并确定该探针的染色体定位。对存在变异探针的相邻基因组区域设置高密度探针阵列。
将单个家系的候选探针设计于单张4×244k或8×60k寡核苷酸微阵列上，开展第二轮筛选。通过更换Cy3与Cy5荧光标记染料，分两轮完成杂交，筛选出存在变异的侧翼探针，并确定其染色体定位。
最后，采用TaqMan®拷贝数检测试剂盒对筛选得到的突变进行验证。