Name: detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice
Uploaded: 2017-01-11T15:00:00.000+00:00
Duration: 10 min 14 s
Description: 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

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?...

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.

<table><tbody><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>221198-100ML</td><td></td></tr><tr><td>SSC Buffer 20&amp;times; Concentrate</td><td>Sigma-Aldrich</td><td>S6639-1L</td><td></td></tr><tr><td>SKY Laboratory Reagent for Mouse</td><td>Applied Spectral Imaging</td><td>FPRPR0030/M40</td><td></td></tr><tr><td>CAD - Concentrated Antibody Detection Kit</td><td>Applied Spectral Imaging</td><td>FPRPR0033</td><td></td></tr><tr><td>Single Paints Customized - 3 Colors; Mouse chromosome 1: Red, Mouse chromosome 2: Green, Mouse chromosome 3: Aqua</td><td>Applied Spectral Imaging</td><td>FPRPR0182/10</td><td></td></tr><tr><td>Glass coverslips</td><td>Fisher Scientific</td><td>12-545B</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-100</td><td></td></tr><tr><td>Hydrochloric acid, 37%, Acros Organics</td><td>Fisher Scientific</td><td>AC45055-0025&amp;nbsp;</td><td></td></tr><tr><td>Fisherbrand Glass Staining Dishes&amp;nbsp; with Screw Cap</td><td>Fisher Scientific</td><td>08-816</td><td></td></tr><tr><td>KaryoMAX Potassium Chloride Solution&amp;nbsp;</td><td>Life Technologies</td><td>10575-090</td><td></td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Colcemid powder</td><td>Fisher Scientific</td><td>50-464-757&amp;nbsp;</td><td></td></tr><tr><td>Histopaque-1083&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10831</td><td></td></tr><tr><td>Shepherd Mark I, model 25&lt;sup&gt; 137&lt;/sup&gt;Cs irradiator</td><td>J. L. Shepherd &amp;amp; Associates</td><td>Model 484B</td><td></td></tr><tr><td>Syringe 1 mL</td><td>BD Biosciences</td><td>647911</td><td></td></tr><tr><td>Ethyl Alcohol, 200 Proof</td><td>Fisher Scientific</td><td>MEX02761</td><td></td></tr><tr><td>PBS, (1x PBS Liq.), w/o Calcium and Magnesium</td><td>Fisher Scientific</td><td>ICN1860454</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10-437-010</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454SK-4</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>AC295320010</td><td></td></tr><tr><td>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。 <p class="jove...

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

Universidad San Sebastian

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 Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

科研

癌症研究

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

医学

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

一种人外周血淋巴细胞γ-H2AX病灶测定的自动显微镜评分方法

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

免疫与感染

鉴定小鼠前期停滞卵母细胞中的 DNA 损伤

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

作者聚焦：了解哺乳动物卵母细胞和植入前胚胎的 DNA 损伤反应 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

检测小鼠卵母细胞中的DNA双链断裂

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

发育生物学

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

通过多色荧光跨染色体畸变稳定的检测

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体内模型对缺氧对肿瘤转移的影响测试

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

一种人外周血淋巴细胞γ-H2AX病灶测定的自动显微镜评分方法

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量