Name: rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish
Uploaded: 2014-08-19T16:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

这项工作是由美国国立卫生研究院资助R00 CA160574（SFB）和由罗格斯大学生物技术培训计划（到SMM）的奖学金支持。

这个过程是经新泽西州的州立大学实验动物管理和使用委员会在罗格斯。小鼠按照美国国立卫生研究院指南实验动物护理和使用处理。科学家应咨询自己的国家和机构的动物组织建立和批准的准则。 开始之前，制备表1中列出的解决方案。 1，B细胞分离和激活安乐死采用的CO 2颈椎脱位鼠标。将鼠标这样身体的左侧是向上和喷鼠标，用70％的乙醇，直至毛发是湿的。解剖脾和地方35毫米组织培养皿用2ml洗涤缓冲液。在层流罩，用5毫升注射器的平头，轻轻的向上突破，在组织培养皿脾。 <ol><li>插入一个70微米的尼龙网成50毫升管和脾脏样品在2毫升的洗涤缓冲液转移到尼龙筛。轻轻地扰乱使用注射器的扁平端的脾脏中的网格。 </li><li>冲洗注射器的后面和35毫米的钢板用8ml的洗涤缓冲液，并通过70微米尼龙筛过滤。离心机在300×g离心在4℃下10分钟。 </li><li>吸出并弃去上清，重悬沉淀物在3ml的ACK裂解缓冲液中。移液器上下简要地破坏团块，并孵育5分钟。加入10毫升的洗涤缓冲液和离心分离机，在300×g离心在4℃下10分钟。 </li><li>吸出并弃去上清液，并通过在1ml的洗涤缓冲液轻敲试管底部，然后通过移液重悬沉淀。加入50μl的抗CD43 MACS微珠并在冰上孵育30分钟。加入10毫升的洗涤缓冲液，轻轻混匀，离心300×g离心4℃10分钟。加入10毫升的洗涤缓冲液，轻轻混匀，离心300×g离心4℃10分钟。 </li><li>设立迷你MACS柱放置一列上的MACS磁铁。定位标记的15毫升锥形管列如下。加入1毫升的洗涤缓冲液对柱，并允许向15ml试管中运行通过。 </li><li>吸去上清，重悬在1ml的洗涤缓冲液。加载样品在柱后的洗涤缓冲液中已运行通过。 </li><li>后的样品通过色谱柱运行，加入1 ml的洗涤缓冲液洗柱。加7毫升的洗涤缓冲液，直接向在15ml试管中收集的样品。 </li><li>计数使用血球细胞。传送/孔所需的500万细胞到50ml管细胞所需的体积。离心机在300×g离心在4℃下10分钟。补充B细胞中与LPS 1:500和IL-4 1:1,000。 </li><li>吸出上清液，并添加所需的百万个细胞/ ml的终浓度添加的B细胞培养基中的适当体积。 </li><li>板的细胞在6孔板中，用5ml的细胞在百万细胞/毫升，将其放入humidifi编细胞培养培养箱中于37℃，5％CO 2。 </li></ol> 2，B细胞固定<ol><li>加入50μl的秋水仙胺的对好含有5ml的B细胞（为100纳克/毫升的终浓度）孵育1小时，在37℃下。 Prewarm 75 mM的KCl溶液在37℃水浴。转移B细胞标记15ml试管中。用胶带和离心标签在300 XG，在4℃下10分钟。 </li><li>吸出上清液留1毫升的管中，通过移液重悬沉淀。加入5毫升预热氯化钾一滴一滴的同时点击或脉冲的旋涡。 </li><li>加入10 ml的KCl和通过反转混合。孵育在37℃水浴15分钟。加入5滴新鲜固定液和混合通过反转。离心机在300×g离心在4℃下10分钟。吸出上清液留1毫升的管，重悬沉淀用移液器上下吹吸。 </li><li>加入5毫升新鲜固定液一滴一滴的同时点击或脉冲在VORTEX。增加一个额外的10毫升固定液和温育30分钟，在室温下进行。 </li><li>离心机在300×g离心在4℃下10分钟。吸出上清液留1毫升管，重悬吹打。 ，增加14毫升新鲜固定液和离心机在300×g离心在4℃下10分钟。 </li><li>重复步骤2.7的两倍以上。 </li><li> ，增加14毫升新鲜固定液。密封瓶盖封口膜过夜存放于-20℃。 </li></ol> 3，制备中期染色体价差<ol><li>设定一个监管环境室22.9°C和52％的湿度。 </li><li>离心机在4℃下固定的B细胞，在300×g离心10分钟。吸出固定液留下70微升的管子，重悬吹打。 </li><li>放置在湿度箱标记的幻灯片以35°的角度。降35微升悬浮B细胞上一张幻灯片，每滴样品两张幻灯片。允许的幻灯片中的湿度室中干燥3​​0分钟，然后保存在幻灯片在37℃的幻灯片框。 </li></ol> 4，端粒PNA FISH <ol><li>探针的制备<ol><li>预温去离子甲酰胺至37℃。拌2微升端粒PNA探针与7微升去离子预热甲酰胺和孵育在37℃下振荡1小时。 </li><li>预温鱼预混至37℃。从步骤4.1.1添加7微升预热的鱼预混到混合物中，在37℃下进行1-3小时。 </li><li>变性的探针混合物8分钟，在80℃。在37℃下预退火的探针1小时。 </li></ol></li><li>染色体幻灯片的预处理<ol><li>预温热含有50ml的0.01M的HCl中和至37℃的水浴中载玻片染色缸。 </li><li>地方载玻片在含有2×SSC 5分钟，在室温下具有不同的载玻片染色缸。 </li><li>加入2微升蛋白酶库存到预热的载玻片染色缸和通过反转混合。传送滑动到该玻璃SLIDE-染色缸和孵化90秒，在37℃。 </li><li>洗涤载玻片2倍于含有1x PBS，每次5分钟，在室温下，振摇不同的载玻片染色缸。然后在室温下清洗载玻片5分钟，在1×PBS / MgCl 2的，摇动。 </li><li>传送的幻灯片到含有新鲜制备的1％甲醛/ 1X PBS / MgCl 2的，在室温下10分钟的载玻片染色缸。 </li><li>为5分钟，在含有1x PBS在室温下具有不同的载玻片染色缸洗载玻片，振摇。 </li><li>由具有含70％，90％和100％的乙醇分离载玻片染色罐制备乙醇脱水系列。 </li><li>传送滑动到每个罐3分钟，开始用70％乙醇，然后90％乙醇中，然后加入100％乙醇。完成后，将乙醇罐在-20℃。 </li><li>通过点击过量的乙醇到纸巾上，并以35度角支撑幻灯片允许的幻灯片风干。干燥后，马RK 18×18毫米面积上用金刚石笔幻灯片的后面杂交。 </li></ol></li><li>变性幻灯片鱼<ol><li>适用于120微升70％去离子甲酰胺/ 2×SSC为24 x 60毫米盖玻片。触摸滑动盖玻片。变性的滑动，在80℃的热板上进行90秒。 </li><li>很快，小心滑出盖玻片，并放置在冰冷的70％乙醇中的幻灯片为3分钟，接着进行90％乙醇和100％乙醇中各3分钟。使载玻片在空气中干燥。 </li><li>由衬塑料盒，密封的盖子，湿纸巾准备湿热试验箱。该文件应该是潮湿，但不饱和水。 </li><li>在潮湿的室内，申请步骤4.1.3预退火探针混合物来标记的区域在幻灯片上，盖上一个18×18毫米盖玻片，并在37℃1小时。 </li></ol></li><li>后孵化淘<ol><li>预温50％甲酰胺/ 2×SSC，1×SSC，以及4x SSC / 0.1％吐温20的45°;水浴。 </li><li>小心取出盖玻片，并在预热的50％甲酰胺/ 2×SSC 3xfor 5分钟洗载玻片每个在黑暗和摇晃。在预热的1×SSC中洗幻灯片3xfor每次5分钟，摇晃。最后，洗玻片在预热的4倍的SSC / 0.1％Tween-20的3xfor每次5分钟。 </li><li>染色玻片3分钟的DAPI在避光载玻片染色缸。 </li><li>洗幻灯片5分钟的2X SSC，颤抖。申请35微升的Mowiol安装介质，覆盖24×60毫米的盖玻片，并存储在幻灯片在4℃下。 </li></ol></li></ol>染色体的5微观分析<ol><li>分析使用的是标准的落射荧光显微镜中期幻灯片。使用一种成像平台与自动阶段如METASYSTEMS Metafer平台，以获得最佳效果。对于每个中期扩展，收集一个图像为DAPI荧光可视化的染色体，并用于从端粒P上的荧光信号的一个图像长袍。 （ 例如 ，Cy3标记的其他荧光剂是可用的，并同样很好地工作。） </li><li>用图像分析程序叠加图像，并分析。 </li></ol>

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	<li>Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., Linn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+mammalian+DNA+repair+and+the+DNA+damage+checkpoints.">Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.</a> Ann Rev Biochem. 73, 39-85 (2004).</li><li>Sperka, T., Wang, J., Rudolph, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+checkpoints+in+stem+cells,+ageing+and+cancer.">DNA damage checkpoints in stem cells, ageing and cancer.</a> Nature reviews Mol Cell Biol. 13 (9), 579-590 (2012).</li><li>Alexandrov, L. B., 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=Signatures+of+mutational+processes+in+human+cancer.">Signatures of mutational processes in human cancer.</a> Nature. 500 (7463), 415-421 (2013).</li><li>Bunting, S. F., 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=53BP1+inhibits+homologous+recombination+in+Brca1-deficient+cells+by+blocking+resection+of+DNA+breaks.">53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.</a> Cell. 141 (2), 243-254 (2010).</li><li>Padilla-Nash, H. M., Barenboim-Stapleton, L., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+analysis+of+human+and+mouse+chromosomes.">Spectral karyotyping analysis of human and mouse chromosomes.</a> Nat Protoc. 1 (6), 3129-3142 (2006).</li><li>Callen, E., 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=Chimeric+IgH-TCRalpha/delta+translocations+in+T+lymphocytes+mediated+by+RAG.">Chimeric IgH-TCRalpha/delta translocations in T lymphocytes mediated by RAG.</a> Cell Cycle. 8 (15), 2408-2412 (2009).</li><li>Boboila, 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=Alternative+end-joining+catalyzes+robust+IgH+locus+deletions+and+translocations+in+the+combined+absence+of+ligase+4+and+Ku70.">Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.</a> Natl Acad Sci USA. 107 (7), 3034-3039 (2010).</li><li>Benn, P., Delach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphocyte+culture+and+chromosome+analysis.">Human lymphocyte culture and chromosome analysis.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Nguyen, H. N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+spreads+and+spectral+karyotyping+of+human+embryonic+stem+cells.">Metaphase spreads and spectral karyotyping of human embryonic stem cells.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Schreck, R. R., Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Chromosome banding techniques. Unit 2, (2001).</li><li>Gilley, 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=DNA-PKcs+is+critical+for+telomere+capping.">DNA-PKcs is critical for telomere capping.</a> Proc Natl Acad Sci USA. 98 (26), 15084-15088 (2001).</li><li>Bolzan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+aberrations+involving+telomeres+and+interstitial+telomeric+sequences.">Chromosomal aberrations involving telomeres and interstitial telomeric sequences.</a> Mutagenesis. 27 (1), 1-15 (2012).</li><li>Bolzan, A. D., Telomeres Bianchi, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interstitial+telomeric+repeat+sequences+and+chromosomal+aberrations.">interstitial telomeric repeat sequences and chromosomal aberrations.</a> Mutat Res. 612 (3), 189-214 (2006).</li><li>Poon, S. S., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Quantitative fluorescence in situ hybridization (Q-FISH). Unit 18 14, (2001).</li><li>Morales, J. 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=53BP1+and+p53+synergize+to+suppress+genomic+instability+and+lymphomagenesis.">53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis.</a> Proc Natl Acad Sci USA. 103 (9), 3310-3315 (2006).</li><li>Halaby, M. J., 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=Synergistic+interaction+of+Rnf8+and+p53+in+the+protection+against+genomic+instability+and+tumorigenesis.">Synergistic interaction of Rnf8 and p53 in the protection against genomic instability and tumorigenesis.</a> PLoS Genet. 9 (1), e1003259 (2013).</li></ol>

作者没有利益冲突。

而活化的B淋巴细胞是特别适合于制备的有丝分裂染色体涂抹物，其它类型的细胞也可使用。 T淋巴细胞有许多共同的B细胞的优点，因为它们可以从脾或淋巴结进行纯化，并具有高的有丝分裂指数，当由生长在含有适当的促细胞分裂剂8中等刺激。是胚胎干细胞（ES细胞），也适合于中期染色体分析9。如果这些都无法使用，成纤维细胞可以使用，虽然用秋水仙胺更长的治疗（ 例?...

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.&#160;Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Reagents</td>
			<td>Comments</td>
			<td>Storage</td>
		</tr>
		<tr>
			<td rowspan="3">B Cell Wash Buffer</td>
			<td>500 ml 1x Hanks Balanced Salt Solution</td>
			<td rowspan="3">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="3">4 &#176;C</td>
		</tr>
		<tr>
			<td>5 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td rowspan="8">B Cell Medium</td>
			<td>418.5 ml RPMI-1640</td>
			<td rowspan="8">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="8">4 &#176;C</td>
		</tr>
		<tr>
			<td>50 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td>5 ml glutamine</td>
		</tr>
		<tr>
			<td>5 ml nonessential amino acids</td>
		</tr>
		<tr>
			<td>5 ml sodium pyruvate</td>
		</tr>
		<tr>
			<td>1.8 ml 14.2 M 2-mercaptoethanol</td>
		</tr>
		<tr>
			<td>5 ml 1M HEPES</td>
		</tr>
		<tr>
			<td>Fixative</td>
			<td>3:1 v/v methanol/glacial acetic acid</td>
			<td></td>
			<td>Make fresh</td>
		</tr>
		<tr>
			<td rowspan="2">0.075 M KCl</td>
			<td>1.395 g KCl</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>250 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">FISH Master Mix</td>
			<td>40 ml 50% Dextran sulfate</td>
			<td rowspan="3">Vortex the solution and leave in a shaking platform overnight</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml 20x SSC</td>
		</tr>
		<tr>
			<td>40 ml sterile distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">70% Formamide/2x SSC</td>
			<td>10 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7 with 1M HCl</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml distilled H2O</td>
		</tr>
		<tr>
			<td>70 ml deionized formamide</td>
		</tr>
		<tr>
			<td rowspan="2">0.01 M HCl</td>
			<td>1 ml 1M HCl</td>
			<td rowspan="2">Adjust to pH 2.0</td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>99 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="2">1x PBS/MgCl2</td>
			<td>50 ml 1M MgCl2</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>950 ml 1x PBS</td>
		</tr>
		<tr>
			<td rowspan="3">50% Formamide/2x SSC</td>
			<td>20 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7.25 
			Formamide used in this step does not have to be deionized.</td>
			<td rowspan="3">Make fresh</td>
		</tr>
		<tr>
			<td>80 ml distilled H2O</td>
		</tr>
		<tr>
			<td>100 ml formamide</td>
		</tr>
		<tr>
			<td rowspan="3">4x SSC/0.1% Tween20</td>
			<td>50 ml 20x SSC</td>
			<td rowspan="3"></td>
			<td rowspan="3">Room Temperature</td>
		</tr>
		<tr>
			<td>200 ml distilled H2O</td>
		</tr>
		<tr>
			<td>250 &#956;l Tween20</td>
		</tr>
		<tr>
			<td rowspan="2">DAPI staining solution (80 ng/ml)</td>
			<td>40 &#956;l 0.2mg/ml DAPI stock</td>
			<td rowspan="2"></td>
			<td rowspan="2">4 &#176;C in light protected staining jar</td>
		</tr>
		<tr>
			<td>100 ml 2x SSC</td>
		</tr>
		<tr>
			<td rowspan="5">Mowiol Mounting Medium</td>
			<td>6 g glycerol</td>
			<td rowspan="5">Incubate at 50 &#176;C with stirring for 2hrs</td>
			<td rowspan="5">Aliquot and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>2.4 g Mowiol 4-88</td>
		</tr>
		<tr>
			<td>6 ml distilled H2O</td>
		</tr>
		<tr>
			<td>12 ml 0.2 M TrisCl pH 8.5</td>
		</tr>
		<tr>
			<td>230 &#956;l 1% Thimerosal (w/v in water)</td>
		</tr>
	</tbody>
</table>
Table 1. List of solutions.&#160;

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.

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

癌症是通过影响基因调节正常细胞生长的突变的积累而引起的。突变是变化所引起的DNA损伤的基因组的结构和序列的结果。 DNA损伤的发生原因，通过各种方法，包括外源试剂如电离辐射，和作为正常细胞的代谢，如通过与活性氧1接触存在的核苷酸碱基或损害自发脱氨的一种副产品。 虽然哺乳动物细胞中具有一系列的维修活动的可逆转的DNA损伤或恢复在断裂位点的序列，突变仍然积累在整个细胞的寿命。 DNA损伤可进一步促进衰老和干细胞的效能损失，其中两个是与衰老相关的疾病相关联的进程。在处理两个符号理解DNA损伤的修复，因此至关重要ificant问题的公众健康。越来越多的证据表明，哺乳动物DNA修复途径可以向肿瘤细胞的基因组3,4的演变，使得它更有必要了解参与抑制突变在分子水平上的处理。 染色体畸变的直接可视化是一个强大的和定量测定特定细胞类型的基因组不稳定性的程度手段。从细胞中冷凝的染色体在分裂中期，可以分离并利用光或荧光显微镜检查。这样的细胞遗传学方法已经在实践了几十年，并且可以用于证明易位或特定类型与DNA修复活性的损失相关联的染色体畸变的出现。该协议适合于一些潜在的扩展:染色体可以标记探针光谱核型分析（SKY）或多色荧光原位杂交（mFISH）确定易位5,6。这些技术也使染色体易位的频率和复杂的染色体易位结构来确定，这超出了能够与本协议提供的附加信息。备选地，序列特异性探针可产生和使用在选择的基因组位点7来测试DNA断裂的频率。 在这个协议中，我们描述了编制中期染色体由B淋巴细胞传播。荧光标记的肽核酸（PNA）探针，端粒重复序列的情况下，其有效标记的端粒在分裂中期染色体扩散该协议具有几个优点。 B细胞可以被诱导生长在高有丝分裂指数，使高品质的差可以持续进行制备。从转基因小鼠的B细胞也不太可能包含次要的基因突变，可以颠倒的贡献的分析特定基因的基因组的完整性。在PNA-FISH方法可以在一天内完成，并允许染色体断裂的更准确的得分。通过使用这种方法，特别是与在此协议中描述的特定设备的组合，所以能够产生非常一致的，高品质的差和快速分析的速率和基因组不稳定性的类型。

<table><tbody><tr><td>50% Dextran sulfate</td><td>Intergen</td><td>S4030</td><td></td></tr><tr><td>Deionized formamide</td><td>Ambion</td><td>9342</td><td></td></tr><tr><td>Pepsin (5 g)</td><td>Sigma</td><td>P 6887&amp;nbsp;</td><td></td></tr><tr><td>FCS</td><td>Gemini</td><td>100-106</td><td></td></tr><tr><td>LPS (100 mg)</td><td>Sigma</td><td>L2630</td><td></td></tr><tr><td>IL-4 (5 &amp;mu;g)</td><td>Sigma</td><td>I1020</td><td></td></tr><tr><td>PNA Telomere Probe</td><td>PNA Bio Inc</td><td>F1002</td><td>(CCCTAACCCTAACCCTAA)</td></tr><tr><td>Colcemid</td><td>Roche</td><td>295892</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma</td><td>81381-50g</td><td></td></tr><tr><td>CD43 (ly-48_) micro-beads</td><td>Miltenyl Biotec.</td><td>130-049-801</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>32670-5MG</td><td></td></tr><tr><td>Eclipse E800</td><td>Nikon</td><td></td><td></td></tr><tr><td>AxioImager.Z2</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CDS-5 Cytogenetic Drying Chamber</td><td>Thermotron</td><td></td><td></td></tr></tbody></table>

rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish

有缺陷的DNA修复导致增加的基因组不稳定性，这是基因突变，导致肿瘤发生的根本原因。在不同类型的细胞的频率和染色体畸变的类型的分析，可以在DNA修复途径缺陷待阐明。理解哺乳动物DNA修复生物学已经由生产小鼠用在特定的基因敲除极大地帮助。此协议的目的是量化小鼠B淋巴细胞的基因组不稳定性。使用PNA-FISH探针（肽核酸-荧光原位杂交）的端粒的标签有利于基因组不稳定性的中期染色体利差的快速分析。 B细胞有相对的成纤维细胞特定的优势，因为他们有正常的染色体倍性和较高的有丝分裂指数。因此，B细胞的短期培养使基因组不稳定性的精确测量在原代细胞群这很可能具有较少的次级基因突变总比什么通常发现在转化的成纤维细胞或患者的细胞系。

从一个细胞衍生中期染色体应形成包含40条染色体（如果使用小鼠细胞）（ 图1A）的离散簇。每个染色体具有着丝点的一端，它是作为一个淡蓝色球体可见。在正常的染色体，2端粒信号被视为在染色单体和两个信号由每个着丝粒的末端。间期核将存在于滑动为好。这些将作为蓝色球体，用红色端粒信号，但是没有稠染色体（例如，参见图1B）中可见。在间期核中，可以忽?...

Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

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

DNA修复途径对于维持基因组的完整性，防止基因突变和癌症至关重要。该协议的目的是通过直接观察染色体畸变的量化基因组不稳定性在中期从原位杂交（FISH）对端粒DNA重复使用荧光小鼠B细胞的扩散。

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 ...

rapid-analysis-chromosome-aberrations-mouse-b-lymphocytes-pna

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

16.9K 次观看.  Rutgers, the State University of New Jersey.  DNA修复途径对于维持基因组的完整性，防止基因突变和癌症至关重要。该协议的目的是通过直接观察染色体畸变的量化基因组不稳定性在中期从原位杂交（FISH）对端粒DNA重复使用荧光小鼠B细胞的扩散。 

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

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. ...

科研

遗传学

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.

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 ...

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

鱼的胚胎植入前基因诊断

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

医学

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

从斑马鱼精子细胞中培养染色体传播

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

斑马鱼神经母细胞瘤转移模型

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

癌症研究

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 ...

生物学

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

从小鼠佩耶的补丁中对Germinal中心B细胞JH4因特龙的体细胞突变分析

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

免疫与感染

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

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使用了大气颗粒物引起的染色体畸变分析

通过多色荧光跨染色体畸变稳定的检测