这项研究是由阿肯色州空间格兰特财团和国家空间生物医学研究所通过美国国家航空和航天局的支持下，授予NNX15AK32A（RP）和RE03701（MH-J），和P20 GM109005（MH-J），以及美国退伍军人管理局（ MH-J）。我们感谢克里斯托弗Fettes，环境和职业健康的在阿肯色大学医学科学部计划协调员，在准备稿件的编辑协助。

所有的动物研究都严格按照该指南中的建议，美国国立卫生研究院的实验动物的护理和使用进行。动物方案经阿肯色大学医学科学机构的动物护理和使用委员会。新鲜空气和向标准啮齿动物食物和自由饮水15小时周期 - 所有动物在20±2℃，10在标准空调动物设施收纳。抵达后，小鼠隔离举行1周，并提供认证的啮齿动物食物。 1.Radiation曝光并在中期细胞的体内逮捕<ol><li>揭露联合国麻醉小鼠2戈瑞全身辐射照射室。在辐照过程中，在通风良好的固定室老鼠的地方，以确保他们不会自由移动，并获得一致的剂量。 </li><li>注入100微升0.05％秋水仙素溶液（c溶解的钙和镁的PBS olchicine粉末）腹膜内使用连接用1mL一次性注射器将25g针。避免直接注射到任何器官。 </li><li>留在笼子里动物的骨髓细胞收获前2小时。观察疼痛或痛苦的任何迹象的动物。 </li></ol> 2.骨髓细胞采收和密度梯度离心骨髓单个核细胞的分离<ol><li>安乐死的CO 2窒息鼠标。 </li><li>喷在背腹侧70％的乙醇。 </li><li>就用锋利的剪刀腹部皮肤2厘米的切口。抓住钝镊子切口两侧皮肤，轻轻拉开腹部肌肉。 </li><li>切下的后腿用剪刀，并立即将它们放置在预冷的PBS，4％FBS。 </li><li>仔细清理附着于后肢用锋利的剃刀刃和棉花纱布包扎的所有肌肉。 </li><li>分离从股骨胫骨。修剪用剃刀刃每个骨骼的两个技巧。 </li><li>冲洗骨髓用3mL的PBS（含4％的FBS）的使用连接到一个3毫升注射器23政针的内容，并收集在15毫升离心管中的内容。通过细胞悬浮液的至少10倍通过针，使单细胞悬浮液。 </li><li>小心覆盖等体积的淋巴细胞分离介质（3mL）中对细胞悬浮液，而不会干扰细胞悬浮液和淋巴细胞分离介质之间的接口。离心在400 xg离心在室温下30分钟。 </li><li>小心收集血沉棕黄层，而不会干扰其它层和传输在新的15毫升离心管中。 </li></ol> 3.中期细胞价差的制备<ol><li>加入10 mL的PBS含有血沉棕黄层的管。 </li><li>离心在400 xg离心在室温下5分钟。 </li><li>小心取出上清液。然后分手Ť他细胞沉淀，轻轻敲击，并添加10毫升的PBS。 </li><li>离心在400 xg离心在室温下5分钟。 </li><li>而不干扰细胞团块除去上清，打散沉淀，并添加4mL的预热低渗0.075 1M氢氯化物溶液。通过添加温和持续振荡下跌低渗溶液滴。 </li><li>孵育细胞20分钟，在37℃的水浴中。 </li><li>低渗处理后，添加固定剂的等体积（4mL）中（甲醇:冰醋酸，3:1体积/体积）中的15毫升管中并通过反相管轻轻混匀。 </li><li>离心在400 xg离心在室温下5分钟并弃上清。 </li><li>分手的细胞沉淀用攻丝接着温和涡流几秒钟，并通过与恒定振荡滴加入3毫升固定剂溶液滴。 </li><li>孵育在室温下30分钟，然后在400 xg离心离心5分钟。 </li><li>除去上清液，分手绅士细胞沉淀乐攻，加3毫升新鲜固定液。 </li><li>离心在400 xg离心在室温下5分钟，并除去上清液。分手轻轻敲击细胞沉淀，并加入新鲜的3毫升固定液。 </li><li>重复步骤3.12两倍多。 </li><li>离心在400 xg离心在室温下5分钟，并除去上清液而不扰乱细胞沉淀。然后加入400〜600μL固定液和吹打调匀。 </li><li>滴30微升固定的细胞上以45°角倾斜的预清洗和湿载玻片并允许滑动到空气完全干燥过夜。 </li></ol>通过渔业部4小鼠染色体绘画<ol><li>染色体变性<ol><li>将包含中期细胞利差2X SSC在室温下2分钟的幻灯片。 </li><li>脱水串行乙醇洗涤滑板为70％每2分钟，80％，和100％。在65℃下孵育60分钟。 </li><li>预热40毫升变性溶液（70％甲酰胺/ 2×SSC; pH值7.0）至70℃（±2℃）下在玻璃科普林缸30分钟。 1.5分钟 - 通过温育在预热的变性溶液滑动1变性染色体。 </li><li>骤立即滑到在冰冷的70％乙醇2分钟，停止变性过程以及防止变性染色体从重新退火。然后，通过用80％乙醇洗涤2分钟脱水。重复用100％乙醇的乙醇洗涤2分钟。完全干燥后在室温下的幻灯片。 </li></ol></li><li>变性和探针混合物杂交<ol><li>短暂离心由制造商提供的探针混合物，在80℃（±2℃）的水浴中传送探测混合液10μL到500μL的卡扣帽离心管中，和变性温育7分钟。 </li><li>将带有探针混合物的离心管在水浴中于37℃10分钟。 </li><li>变性探针混合物应用到与变性chromoso幻灯片MES。 </li><li>仔细覆盖18毫米×18毫米的玻璃盖玻片的区域以及非常轻轻按压盖玻片滑动消除任何可见的气泡。密封用橡胶水泥盖玻片的所有四个侧面和在用于杂交的潮湿室中在37℃孵育12小时，在黑暗中16小时。 </li></ol></li><li>杂交后清洗和检测<ol><li>小心取出橡胶水泥和盖玻片。 </li><li>在74℃（±2℃）下5分钟在预温0.4X SSC地方滑动。 </li><li>在2分钟的洗涤溶液II（4×SSC / 0.1％吐温-20）洗涤滑动。 </li><li>放置用DAPI复染（1.5微克/毫升）的抗褪色封固剂的20微升，用玻璃盖玻片覆盖。轻轻按下与实验室组织盖玻片，以去除气泡和多余的安装解决方案。用指甲油密封盖玻片的边缘。 </li><li>使用配备有适当的过滤器的荧光显微镜视图幻灯片。 </li></ol></l我&gt; </ol> 5.光谱核型分析小鼠染色体的（SKY） <ol><li>染色体和探针变性<ol><li>平衡滑动在2×SSC在室温下进行2分钟，不摇动。 </li><li>脱水在乙醇系列（100％乙醇70％，80％，和），在室温下每2分钟幻灯片。空气干燥幻灯片完全去除乙醇。 </li><li>在玻璃科普林缸30分钟暖40毫升变性溶液（pH 7.0 70％甲酰胺/ 2×SSC）。 </li><li>孵育在1至1.5分钟预热的变性溶液滑动。 </li><li>立即浸入滑动入冰冷的70％乙醇2分钟，停止变性过程以及防止变性染色体从重新退火。 </li><li>通过在室温下将其放入80％乙醇中2分钟，然后为100％乙醇2分钟，脱水的幻灯片。 </li><li>在设定为80℃（±2℃）水浴变性SKY探头（瓶＃1由制造商提供）7分钟，然后立即放入设定为37℃，10分钟的不同水浴中。 </li></ol></li><li>探针杂交<ol><li>添加变性SKY探针的10微升到变性染色体。 </li><li>小心地将18毫米×18毫米的玻璃盖玻片冲上天空探头，以便没有气泡盖玻片被困。 </li><li>密封用橡胶水泥盖玻片的边缘。 </li><li>放置幻灯片在加湿遮光室并在37℃下在黑暗中孵育24小时至36小时。 </li></ol></li><li>杂交后的洗涤和荧光检测<ol><li>不干扰盖玻片非常小心取出橡皮泥。 </li><li>放置滑入预热快速洗涤液（0.4倍SSC）在72℃（±2℃）下5分钟，以恒定振荡。沉浸滑入洗涤溶液III（4×SSC / 0.1％吐温20）和孵育1分钟，同时振摇。 </li><li>可选:加入80微升阻断剂的（小瓶＃2由制造商提供的）上的杂交的区域中，放置一个24毫米×60毫米的塑料盖玻片，并在37℃下在黑暗中温育30分钟，在湿润的腔室中。 </li><li>轻轻取出塑料盖玻片和洗涤用预热（45℃）洗涤溶液III滑动5分钟。 </li><li>应用80 Cy5信号μL染色试剂（瓶＃3制造商提供），放置24毫米×60毫米的塑料盖玻片，在黑暗中，在加湿室内37℃下孵育40分钟。 </li><li>浸滑入含有玻璃科普林缸预热（45℃）洗涤溶液III（4×SSC / 0.1％吐温20），并在水浴中于45℃孵育2分钟，振摇。重复此洗涤步骤3次。 </li><li>应用80经Cy5.5的μL染色试剂（瓶＃4由制造商提供），放置24毫米×60毫米的塑料盖玻片，在黑暗中，在加湿室内37℃下孵育40分钟。 </li><li>与预热洗幻灯片3次（45℃）洗涤液III。 </li><li>持反对纸巾倾斜位置排出多余液体的幻灯片。添加20μL的抗褪色DAPI试剂（小瓶＃5由制造商提供），并小心地将24毫米×60毫米的玻璃盖玻片而不引入任何气泡。密封盖玻片的边缘与指甲油和与配备用于捕获SKY图像的落射荧光显微镜观察。 </li></ol></li></ol>

<ol>
	<li>Kodama, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+chromosome+aberrations+in+atomic+bomb+survivors:+results+from+25+years+of+investigation.">Stable chromosome aberrations in atomic bomb survivors: results from 25 years of investigation.</a> Radiat Res. 156, 337-346 (2001).</li><li>Lucas, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+signature+for+ionizing+radiation.">Cytogenetic signature for ionizing radiation.</a> Int J Radiat Biol. 73, 15-20 (1998).</li><li>Zaccaria, A., Barbieri, E., Mantovani, W., Tura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+radiation-induced+aberrations+in+patients+with+Hodgkin's+disease.+Possible+correlation+with+second+malignancy?.">Chromosome radiation-induced aberrations in patients with Hodgkin's disease. Possible correlation with second malignancy?.</a> Boll. Ist. Sieroter. Milan. 57, 76-83 (1978).</li><li>Fisher, N. L., Starr, E. D., Greene, T., Hoehn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+and+limitations+of+chromosome+banding+in+pre-+and+postnatal+service+cytogenetics.">Utility and limitations of chromosome banding in pre- and postnatal service cytogenetics.</a> Am J Med Genet. 5, 285-294 (1980).</li><li>Hande, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+chromosome+aberrations+persist+in+individuals+many+years+after+occupational+exposure+to+densely+ionizing+radiation:+an+mFISH+study.">Complex chromosome aberrations persist in individuals many years after occupational exposure to densely ionizing radiation: an mFISH study.</a> Genes Chromosomes. Cancer. 44, 1-9 (2005).</li><li>Gall, J. G., Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+detection+of+RNA-DNA+hybrid+molecules+in+cytological+preparations.">Formation and detection of RNA-DNA hybrid molecules in cytological preparations.</a> Proc Natl Acad Sci U S A. 63, 378-383 (1969).</li><li>Bauman, J. G., Wiegant, J., Borst, P., van, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+fluorescence+microscopical+localization+of+specific+DNA+sequences+by+in+situ+hybridization+of+fluorochromelabelled+RNA.">A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA.</a> Exp Cell Res. 128, 485-490 (1980).</li><li>Hopman, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-color+detection+of+two+target+DNAs+by+non-radioactive+in+situ+hybridization.">Bi-color detection of two target DNAs by non-radioactive in situ hybridization.</a> Histochemistry. 85, 1-4 (1986).</li><li>Nederlof, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-color+fluorescence+in+situ+hybridization+for+the+simultaneous+detection+of+multiple+nucleic+acid+sequences.">Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences.</a> Cytometry. 10, 20-27 (1989).</li><li>Nederlof, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+fluorescence+in+situ+hybridization.">Multiple fluorescence in situ hybridization.</a> Cytometry. 11, 126-131 (1990).</li><li>Szeles, A., Joussineau, S., Lewensohn, R., Lagercrantz, S., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+spectral+karyotyping+(SKY)+in+biodosimetry+for+the+triage+situation+following+gamma+irradiation.">Evaluation of spectral karyotyping (SKY) in biodosimetry for the triage situation following gamma irradiation.</a> Int J Radiat Biol. 82, 87-96 (2006).</li><li>Camparoto, M. L., Ramalho, A. T., Natarajan, A. T., Curado, M. P., Sakamoto-Hojo, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+analysis+by+the+FISH-painting+method+for+retrospective+dose+reconstruction+in+individuals+exposed+to+ionizing+radiation+10+years+after+exposure.">Translocation analysis by the FISH-painting method for retrospective dose reconstruction in individuals exposed to ionizing radiation 10 years after exposure.</a> Mutat. Res. 530, 1-7 (2003).</li><li>Edwards, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critique+of+'Collaborative+exercise+on+the+use+of+FISH+chromosome+painting+for+retrospective+biodosimetry+of+Mayak+nuclear-industrial+personnel.">A critique of 'Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.</a> Int J Radiat Biol. 78, 867-871 (2002).</li><li>Bauchinger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+exercise+on+the+use+of+FISH+chromosome+painting+for+retrospective+biodosimetry+of+Mayak+nuclear-industrial+personnel.">Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.</a> Int J Radiat Biol. 77, 259-267 (2001).</li><li>Hieber, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+rearrangements+in+post-Chernobyl+papillary+thyroid+carcinomas:+evaluation+by+spectral+karyotyping+and+automated+interphase+FISH.">Chromosomal rearrangements in post-Chernobyl papillary thyroid carcinomas: evaluation by spectral karyotyping and automated interphase FISH.</a> J Biomed. Biotechnol. 2011, 693691 (2011).</li><li>Cohen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=detection+of+chromosome+rearrangements+in+two+cases+of+tMDS+with+a+complex+karyotype.">detection of chromosome rearrangements in two cases of tMDS with a complex karyotype.</a> Cancer Genet Cytogenet. 138, 128-132 (2002).</li><li>Pathak, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vitamin+E+Analog+Gamma-Tocotrienol+(GT3)+Suppresses+Radiation-Induced+Cytogenetic+Damage.">The Vitamin E Analog Gamma-Tocotrienol (GT3) Suppresses Radiation-Induced Cytogenetic Damage.</a> Pharm Res. , (2016).</li><li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+chromosome+spreading.">Dynamics of chromosome spreading.</a> Am J Med Genet. 61, 387-393 (1996).</li><li>Deng, W., Tsao, S. W., Lucas, J. N., Leung, C. S., Cheung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+improving+metaphase+chromosome+spreading.">A new method for improving metaphase chromosome spreading.</a> Cytometry A. 51, 46-51 (2003).</li><li>Hande, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past+exposure+to+densely+ionizing+radiation+leaves+a+unique+permanent+signature+in+the+genome.">Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome.</a> Am J Hum Genet. 72, 1162-1170 (2003).</li><li>Tug, E., Kayhan, G., Kan, D., Guntekin, S., Ergun, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+long-term+effects+of+ionizing+radiation+through+measurement+of+current+sister+chromatid+exchange+(SCE)+rates+in+radiology+technologists,+compared+with+previous+SCE+values.">The evaluation of long-term effects of ionizing radiation through measurement of current sister chromatid exchange (SCE) rates in radiology technologists, compared with previous SCE values.</a> Mutat. Res. 757, 28-30 (2013).</li><li>Kanda, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fluorescent+chromosome+painting+using+the+peroxidase/diaminobenzidine+(DAB)+reaction.">Non-fluorescent chromosome painting using the peroxidase/diaminobenzidine (DAB) reaction.</a> Int J Radiat Biol. 73, 529-533 (1998).</li><li>Kakazu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+spectral+colour+banding+in+cytogenetic+analysis.">Development of spectral colour banding in cytogenetic analysis.</a> Lancet. 357, 529-530 (2001).</li><li>Rithidech, K. N., Honikel, L., Whorton, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mFISH+analysis+of+chromosomal+damage+in+bone+marrow+cells+collected+from+CBA/CaJ+mice+following+whole+body+exposure+to+heavy+ions+(56Fe+ions).">mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (56Fe ions).</a> Radiat Environ Biophys. 46, 137-145 (2007).</li><li>Abrahams, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+FISHing+of+transgenic+mice+recommended:+FISH+and+SKY+define+BAC-mediated+balanced+translocation.">Metaphase FISHing of transgenic mice recommended: FISH and SKY define BAC-mediated balanced translocation.</a> Genesis. 36, 134-141 (2003).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+scoring+of+FISH-&amp;#34;painted&amp;#34;+chromosome-type+exchange+aberrations.">On the scoring of FISH-&amp;#34;painted&amp;#34; chromosome-type exchange aberrations.</a> Mutat. Res. 307, 345-353 (1994).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+"painting"+patterns+resulting+from+complex+exchanges.">FISH "painting" patterns resulting from complex exchanges.</a> Mutat. Res. 312, 51-60 (1994).</li><li>Tucker, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAINT:+a+proposed+nomenclature+for+structural+aberrations+detected+by+whole+chromosome+painting.">PAINT: a proposed nomenclature for structural aberrations detected by whole chromosome painting.</a> Mutat. Res. 347, 21-24 (1995).</li><li>Knehr, S., Zitzelsberger, H., Bauchinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH-based+analysis+of+radiation-induced+chromosomal+aberrations+using+different+nomenclature+systems.">FISH-based analysis of radiation-induced chromosomal aberrations using different nomenclature systems.</a> Int J Radiat Biol. 73, 135-141 (1998).</li><li>Lucas, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+translocation+frequency+analysis+in+humans+decades+after+exposure+to+ionizing+radiation.">Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation.</a> Int J Radiat Biol. 62, 53-63 (1992).</li><li>Braselmann, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKY+and+FISH+analysis+of+radiation-induced+chromosome+aberrations:+a+comparison+of+whole+and+partial+genome+analysis.">SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis.</a> Mutat. Res. 578, 124-133 (2005).</li><li>Sokolova, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+multitarget,+multicolor+fluorescence+in+situ+hybridization+assay+for+the+detection+of+urothelial+carcinoma+in+urine.">The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine.</a> J Mol Diagn. 2, 116-123 (2000).</li><li>Lindholm, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodosimetry+after+accidental+radiation+exposure+by+conventional+chromosome+analysis+and+FISH.">Biodosimetry after accidental radiation exposure by conventional chromosome analysis and FISH.</a> Int J Radiat Biol. 70, 647-656 (1996).</li></ol>

The authors declare that they have no competing financial interests.

几个关键步骤，确定渔业部和天空的成功。第一个也是最关键的一步是优化体内骨髓单个核细胞有丝分裂逮捕秋水仙碱治疗。秋水仙素浓度和处理时间是单独或共同确定有效的染色体绘画有丝分裂指数以及染色体浓缩，两个重要的先决条件。高浓度的秋水仙素或更长的治疗时间导致高度浓缩的染色体，不兼容的适当变性和杂交。此外，与冷凝染色体流传中期细胞复合重排的准确识别并不是一件容易的任务来实现。在22和26 g的体重的小鼠100微升0.05％秋水仙碱2小时的腹膜内施用产生中期细胞的足够数量的具有适度冷凝染色体传播。第二个关键步骤是低渗处理。量，处理时间，和低渗溶液影响染色体形态的温度并到达载玻片染色体蔓延。对于更短的时间或在一个亚最佳温度低渗治疗可以防止相应的染色体扩展，导致与重叠染色体传播中期细胞。染色体重叠原位杂交过程中，荧光信号的特异性，和重排的正确标识下游干扰。 第三，后低渗固定是避免细胞团的形成的重要一步。固定剂应该缓慢加入向下以恒定轻轻摇动，以获得单细胞悬浮液的离心管的一侧。否则，中期细胞的利差将成为被困在团块和染色体蔓延将受到损害。然而，染色体也扩展依赖于细胞类型，温度和在T相对湿度滴加细胞悬液到玻片上的输入法。先前的研究由Spurbeck报道，淋巴细胞，60％的相对湿度和20℃的温度下带来更高的中期区18。此外，干燥的持续时间也是最佳染色体扩频的关键步骤。中期细胞扩散的干燥过快产生紧张利差有许多重叠的染色体，而在破中期非常缓慢的干燥效果染色体18。然而，邓表明染色体蔓延，主要取决于相对湿度为各种培养细胞19。在第四个重要的步骤是优化染色体变性时间。更长和更短的时间变性探针杂交产生不利影响。较长的变性导致"浮肿"的染色体，而较短的变性阻碍对染色体探头的互动。减少染色体"浮肿"的另一种方法是对年龄载玻片在室温下过夜是脱颖而出变性步骤。不用于染色体涂染载玻片立即，可以无限期地在-20 C在干燥器中储存。染色体变性时间需要与滑动年龄，围绕中​​期细胞差细胞质的量，并且在该细胞滴到载玻片湿度标准化。探针变性和杂交也是成功的涂染重要。应避免探头明亮的光线直接照射，使荧光信号不输的信号强度。 这是完善的渔业部和天空是用于检测染色体间的稳定的畸变强有力的技术。任何重排以及损失或绘染色体增益可以容易地通过这两种方法来鉴定。然而，内臂和臂间的染色体交换检测由这些技术是不可能的。内臂和臂间的交流染色体鉴定，现在都成为了可能贝科使用，分别特定染色体臂多色FISH探针和mBAND FISH探针的发展。对前核工人先前的研究已经表明，mBAND分析是一个强大的工具，以确定辐射损伤20独特的签名。此外，姐妹染色单体交换的识别，辐射损伤21的一个特征，是不可能用这些技术。此外，这两个渔业部和SKY有限油漆染色体的着丝粒区，这可能导致在双着丝粒染色体易位的误得分能力。该技术也不适于识别易位的确切断点定位。此外，荧光信号不能被永久保存，信号强度随时间减小，和荧光染色体涂染不适合由自动系统的分析。然而，荧光信号强度可以通过存储滑动保存的时间更长的时间ES在冷冻机。 染色体绘画两种改进方法已经发展到克服渔业部和天空的限制:分别是，非荧光涂染22和光谱色条带（SCAN）23。非荧光染色体绘画用在鱼画中的过氧化物酶/二氨基联苯胺（DAB）的优点包括:精确识别着丝粒区域，鉴定染色体重排由亮场显微镜，以及非荧光信号被永久保存和图像分析可以实现自动化。另一方面，由于扫描探针从特定频带的基因组DNA序列制备的染色体特异性，因此，能够准确地识别在染色体带的原点的扫描结束的SKY有利的。 SCAN也便于帧内染色体交换的识别。考虑的改进的技术的优点，这些应视为常规使用我ñ临床细胞遗传学检测。然而，制备由每个染色体对特定频带的基因组DNA序列的探针在技术上是挑战性的，并且限制在分子细胞遗传学其使用。 确定治疗组之间的差是否具有统计显著与否是分子遗传学的另一个重要方面。得分的标准，如中期细胞的数目价差计数，每个处理组，每次分析中传播中期细胞染色体计数使用的动物的数量，所用的命名法来分类像差染色体涂染后观察等中确定的统计不确定性发挥关键作用治疗组之间。虽然各个实验室拥有专有的评分标准，100至300和20个中期细胞在至少3至5只小鼠40以及分隔染色体扩散得分一般建议用于确定在渔业部和SKY肛门统计意义ysis，分别为24，25。此外，命名法的用于在涂染色体分类像差的选择是用于统计评估的不确定性的一个重要参数。目前正在使用的两种流行的命名系统来描述整个染色体涂染探针检测畸变:（1）S＆S分类方法由野人和辛普森26，第27和（2）涂料命名由塔克等人。 28。每个系统都依赖于不同的先决条件结构性染色体畸变的分类都有自己的优点和缺点。标S系统主要适用于像差原籍机理的解释，而涂料提供的异常绘画图案的描述。通过Knehr 等人的研究。在FISH绘染色体辐射诱发染色体畸变指示该涂料的方法，如S＆S系统，也可以被用来确定与一些modificati像差原点在评分标准和将是实际应用29最有用的。 双方渔业部和SKY方法都有各自的局限性。例如，渔业部允许快速得分少训练，但是，它并不筛选所有染色体用于测量像差次数揭示只是局部畸变率，根据在研究中使用的涂染色体的数目。但是，整条染色体观察绘画交流总量的一小部分可以通过最初由卢卡斯等人假设的公式估算。 30和由Braselmann 等进一步改性。 31。这个数学公式认为画的染色体中并涂上与复染染色体之间的交流。例如，让我们在fp表示由涂染色体覆盖的基因组的分数总和1（1 F），图2（f 2）和3（F 3），磨片重新˚F1 = 0.0696中，f 2 = 0.0649和f 3 = 0.0570，该值代表个人染色体的雌性小鼠的基因组的内容。因此， 在fp =（F 1 + F 2 + F 3）= 0.192和复染率变为（1 - 在fp）= 0.808。涉及涂和复染染色体（F P）的交流的频率可以很容易地由二项展开式的叉积（P + Q）来计算2 = P 2 + 2PQ + Q 2，其中p = F p和q =（1 - 在fp）。因此， 男，P = 2PQ = 2F P（1 - 在fp）= 0.310，这意味着染色体交换的31％是画小鼠染色体-1，-2后可观察到的，和-3。因此，1 / 0.31 = 3.23细胞需要被评分到等于一带状中期细胞或一种全基因组当量。此外，无论是渔业部和天空都仅限于特异性的能力ically确定哪些基因和断点都参与了像差形成和还检测小重复，倒置和缺失。 渔业部和SKY分析的应用不仅限于基础研究。两者都经常被在临床研究中使用。这些技术也被应用到产前和产后诊断，风险评估，和不同类型的癌症的鉴别。而且，无论是技术可用于油漆相间染色体。索科洛娃用于渔业部作画相间染色体的第一次32。这些技术也可用于在意外或故意曝光后人类辐照后剂量估算和风险评估电离辐射11，33。因此，通过渔业部或天空的部分或整个基因组的分析，分别是有用的工具，以检测染色体间稳定像差各种研究。

The two most reliable methods of identifying radiation-induced inter-chromosomal stable aberrations are multiple fluorescence in situ hybridization (mFISH), which allows the painting of two or more chromosomes simultaneously, and spectral karyotyping (SKY), which imparts a distinct color to each homologous chromosome pair in the genome. Unlike unstable aberrations, stable aberrations are persistent in nature and may be propagated for several generations in irradiated populations1, and are regarded as critical molecular "signatures" of radiation-induced cytogenetic lesions2. Studies by various groups have shown that stable aberrations are associated with the pathogenesis and development of a number of diseases, including cancer3. Before the era of chromosome painting (also referred as molecular cytogenetics), the conventional G-banding technique was the only method for detecting stable chromosomal aberrations. However, chromosome banding is a challenge to cytogeneticists because the resolution is limited, reproducibility is uncertain, it is a labor-intensive procedure, and it requires highly skilled and experienced cytogeneticists for reliable data interpretation4. Moreover, the classic banding technique does not allow detection of complex chromosomal rearrangements, which involve the interaction of three or more breaks distributed among two or more chromosomes, a common outcome of radiation damage. Complex aberrations may persist in individuals many years after radiation exposure, making them useful for retrospective biodosimetry5. Therefore, an alternate approach was required to overcome the limitations of conventional banding techniques to detect stable chromosomal rearrangements. 
In the late 1960s, the pioneering work of Gall and Pardue (1969) on molecular hybridization using nucleic acid probes labeled with radioactive material commenced a new era in the field of cytogenetics, which allowed detection of a specific DNA sequence on chromosomes6. However, the use of radioactive probes for molecular hybridization had several drawbacks: radioactive probes are relatively unstable, probe activity depends on radioactive decay of the isotope used, hybridization takes a longer time, the resolution is limited, the probes are relatively costly, and the radioactive materials are a health hazardous. Thus, it became necessary to develop and design non-radioactive probes. The introduction of fluorescent tagged nucleic acid probes in the 1980s and 1990s overcame the limitations of radioactive probes and greatly enhanced the safety, sensitivity, and specificity of the hybridization technique7-10. Fluorescent probes give rise to extremely bright signals when observed under fluorescence microscopes equipped with the appropriate excitation and emission filters. Any loss, gain, or rearrangement of fluorescent labeled chromosome/s or a part of the chromosome is easily identifiable with this FISH technique.
Analysis of chromosomal aberrations by FISH painting has led to marked progress in cytogenetic research over the years. Designing fluorescent labeled probes for specific applications ranging from locus-specific probes to whole-chromosome painting probes has advanced the field significantly; this has also facilitated the detection of submicroscopic ("cryptic") rearrangement, which was not possible by conventional chromosome banding. Chromosome painting by mFISH and SKY have proven to be valuable tools for the identification of simple and complex inter-chromosomal rearrangements. The basic principles for both techniques are similar, but the method of detection and discrimination of fluorescent signal after in situ hybridization and the process of image acquisition are different. In mFISH, separate images of each of the four fluorochromes are captured by using narrow bandpass microscope filters; dedicated software is then used to combine the images. While in SKY, image acquirement is based on a combination of epifluorescence microscopy, charge-coupled device imaging, and Fourier spectroscopy, which allows the measurement of the entire emission spectrum with a single exposure at all image points. In both mFISH and SKY, monochrome images are captured independently, then merged, and finally, unique pseudo-colors are assigned to the chromosomes in monochromatic images based on the specific dye attached to each fluorochrome probe.
The contribution of mFISH and SKY analysis in the radiation biology field is remarkable, particularly for retrospective dose estimation of human exposure to IR (radiation biodosimetry)11-14, radiation carcinogenesis risk assessment15, as well as detection and risk estimation of radiotherapy-related secondary cancer16. A recent study on mice has shown that a FISH-based chromosome painting technique is also an important tool for evaluating the efficacy of radiation countermeasure17. In the present study, the effect of total body radiation exposure on the induction of stable chromosomal aberrations in the bone marrow cells of mice has been demonstrated using mFISH and SKY techniques.

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			<td>FPRPR0030/M40</td>
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			<td>Fisher Scientific</td>
			<td>12-545B</td>
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			<td>Fisher Scientific</td>
			<td>BP337-100</td>
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			<td height="20" style="height:20px;">Hydrochloric acid, 37%, Acros Organics</td>
			<td>Fisher Scientific</td>
			<td>AC45055-0025&#160;</td>
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			<td>Fisher Scientific</td>
			<td>08-816</td>
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			<td>Life Technologies</td>
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			<td height="20" style="height:20px;">Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
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			<td>J. L. Shepherd &#38; Associates</td>
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			<td>BD Biosciences</td>
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			<td height="20" style="height:20px;">Ethyl Alcohol, 200 Proof</td>
			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Fisher Scientific</td>
			<td>A454SK-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>AC295320010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss Microscope</td>
			<td>Zeiss</td>
			<td>AXIO Imager.Z2</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

全身照射诱导照射小鼠的骨髓细胞大量染色体畸变。当前协议是用于治疗辐射暴露后在体内的骨髓细胞的有丝分裂停滞优化，从照射小鼠，骨髓单核细胞的分离的后腿骨髓细胞的收获通过密度梯度离心，制备中期细胞价差，以及随后的检测由渔业部和SKY技术辐射诱导稳定染色体畸变。染色体画使用这些技术可用于检测和评估各种病理生理条件的危险。 图1示出了代表中期细胞与正常和异常小鼠染色体对-1，-2和-3传播。 图2显示正常和异常小鼠中期染色体的光谱核型分析。这些结果表明，电离辐射的增殖鼠骨髓细胞，其主要是由该负责自我更新和分化成不同细胞类型的干细胞和祖细胞的诱导染色体间稳定像差。在干细胞和祖细胞的细胞遗传学改变已显示与各种临床疾病，包括癌症相关联，并且被认为是疾病的严重程度和总生存期的最强预测。因此，它是符合逻辑的推断电离辐射诱导的稳定间染色体畸变可能导致癌症的发展的风险增加。 <img alt="图1" src="/files/ftp_upload/55162/55162fig1.jpg" /> 图1:为检测通过渔业部在照射小鼠骨髓细胞辐射的影响。 A和B显示具有正常成对声道的小鼠中期细胞传播romosome-1（红），染色体2（绿色），以及染色体-3（水性）。所有其他染色体复染用DAPI（蓝色）。 C显示涉及染色体-1，染色体1的一部分已被集成在非画（DAPI）染色体，而另一部分已经形成了一个偏心片段稳定像差。 D显示染色体2的一部分的一部分已被集成在非画染色体。 E表示涉及染色体3稳定像差。 F显示涉及所有三个画染色体稳定的畸变。断点和色结由箭头指示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55162/55162fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55162/55162fig2.jpg" /> 图2: 辐射对骨马尔影响辐照鼠标流细胞检测由SKY。上图显示C57BL / 6雌性小鼠的正常光谱核型分析（SKY）。中间面板显示涉及染色体4和染色体12上的稳定的象差。较低的面板显示了涉及染色体7和染色体10一个稳定的染色体畸变鼠标光谱核型分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55162/55162fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

detection-inter-chromosomal-stable-aberrations-multiple-fluorescence

Nanyang Technological University

Research

JoVE Journal

Genetics

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

9.8K 次观看.  University of Arkansas for Medical Sciences. The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation. 

视频: Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

科研

遗传学

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.