我们感谢谢尔盖·德敏和塔季扬娜·Karamysheva他们的帮助与染色体的制备和嗜鱼。我们也感谢为我们提供了白纹伊蚊和致倦库蚊基因组DNA BAC克隆和Melissa韦德的文字编辑大卫Severson的。这项工作得到了两笔赠款，由美国国立卫生研究院：1R21 AI88035-01玛丽亚五Sharakhova的1R21 AI094289-01伊戈尔·Sharakhov。

1。染色体的制备 蚊子幼虫饲养按蚊研究在疟疾研究和参考试剂资源中心的网站中描述的方法使用标准的协议 （MR4）13。饲养蚊子的温度进行了修改，以提供最高的染色体数目成虫盘和幼虫的死亡率最低。蚊子幼虫的发育阶段乃根据自己的头上胶囊13的大小。 <ol><li>哈奇蚊卵在28°C，后2-3天，转让2 次或3龄幼虫至16°C AE。埃及伊蚊和CX。致倦库蚊和为22°C。冈比亚。 </li><li>发布第 4龄幼虫几分钟用于固定在冰上。 </li><li>冷低渗溶液（下降0.5％，因此，将幼虫到幻灯片培养基柠檬酸或0.075 M氯化钾），并将其放置在立体显微镜下。 </li><li>选择椭圆形标识（ 图1B）为进一步解剖的幼虫。 </li><li>斩首幼虫，并减少角质层的幼虫胸部腹面用解剖剪（ 图2A）。额外的切割，在第二或第三腹节，从幼虫的肠道解剖。箭头所示的方向的削减。 </li><li>打开角质层，去除内脏和脂肪体从幼虫。低渗溶液中取出用滤纸的幻灯片，并添加一个新的下降，低渗溶液直接的ID（ 图2B）。使幼虫在低渗溶液中10分钟，在室温下。 </li><li>删除低渗溶液，使用滤纸，并应用卡诺的溶液（3:1的比例的乙醇/乙酸）。加入固定液后，IDS立即变成了白色，并成为很容易在显微镜下可见（ 图2C </s仲&gt;）。 </li><li>用解剖针，删除ID从幼虫（ 图2D），然后将其转移到下降了50％丙酸。 ，从幻灯片中删除任何其他组织，如肠和脂肪体。封面标识与一个unsiliconized的22X22盖玻片，并在室温下保持10分钟。 </li><li>量的滑动用滤纸，及壁球通过轻敲铅笔的橡皮擦的周边上的盖玻片的组织。 </li><li>简单分析使用相差显微镜在100倍或200倍的放大倍率（ 图3）滑动的质量。可以考虑准备与50号染色体利差适合鱼类。 </li><li> DIP和保存在液氮中的幻灯片，直到它停止冒泡。删除从滑动用剃刀刀片，盖玻片和立即的幻灯片转移到一个容器中的70％的乙醇于-20℃下冷冻储存在4℃下进行至少1小时，最好的脱水结果（如果有必要，可以在幻灯片被存储在t他的步骤从几分钟到几天）。 </li><li>脱水幻灯片在一系列的乙醇（70％，80％，100％），在4℃下，每次5分钟，并在室温空气干燥。 </li><li>之前利用它们为鱼，干片存放在-20°C。 </li></ol> 2。提取的DNA重复分数 从 AE 染色体上的BAC克隆DNA探针FISH执行 。埃及伊蚊和 CX。倦需要使用未标记的重复DNA组分阻断非特异性杂交的DNA重复序列的染色体。裂成件几百个bp的单链DNA的重连接如下的C 0 t曲线，其中C 0是初始的单链DNA的浓度，和t是再退火时间。 DNA组分与C0T值等于10 -4 -10 -1或10°-10被认为是中度和高度重复。 <ol><li>提取400-500微克使用Qiagen公司，血液和细胞培养Maxikit从整个成蚊的基因组DNA，并准备100-1,000 ng /μl的DNA溶液在1.2×SSC。 </li><li>使DNA变性成一个加热块与基因组DNA通过将一个安全锁管预热至120℃下进行2分钟。高温度范围为200〜500 bp的片段，DNA。 </li><li>根据DNA浓度，重新关联通过将所述管在60℃下为15-150分钟，，获得C0 吨 DNA馏分至C 0 t3的（表1）的DNA。 </li><li>将所述管与DNA在冰上2分钟。 </li><li>的DNA转移至42°C，加入预热的10倍的S1核酸酶缓冲液和S1核酸酶至最终浓度为100 U每1毫克的DNA，并孵育1小时。 </li><li>通过加入0.1体积的3M乙酸钠和1倍体积的异丙醇，在RT，沉淀DNA。 </li><li>离心14,000 rpm，20分钟，在4℃下</li><li>洗净DNA在70％乙醇中，并再次以14,000 rpm离心10分钟在4℃下</li><li>空气干燥，在TE缓冲液溶解DNA沉淀。 </li><li>测量DNA浓度，通过凝胶电泳和可视化。一般的重复DNA馏分的最终数量表示的原始DNA量的35-50％。 </li></ol> 3。 DNA探针标记 BAC克隆的标记的DNA探针和IGS rDNA探针被用于两个不同的协议。 3.1 BAC克隆标记缺口翻译<ol><li>提取BAC克隆DNA的BAC文库，用Qiagen大构造工具包。 </li><li>准备反应混合物在冰上的缺口转译的标签，最终体积为50μl：1微克分离BAC克隆DNA，0.05 mM的每一个未标记的dATP，dCTP和dGTP和0.015 mM的dTTP的1微升的Cy3-dUTP标记（或另一个荧光染料），0.05毫克/毫升BSA，5微升10倍的缺口转译缓冲液，20 U的DNA聚合酶I和U的DNA酶0.0012。 </li><li>在15℃下孵育20.5小时。 </li><li>加入1μl的0.5M EDTA终止反应。 </li><li>在-20℃下，于黑暗处专区探针。 </li></ol> 3.2 IGS rDNA序列标签使用PCR <ol><li>与最终体积为50微升：200ng的基因组DNA，未标记的dATP，dCTP和dGTP各0.05毫米; 0.015 mM的dTTP的1微升的Cy3-dUTP标记（或另一种荧光染料），5μl的准备反应混合物在冰10倍的PCR-缓冲液; 50 pmol的前锋;联合国（GTGTGCCCCTTCCTCGATGT）和反向;的GA（CTGGTTTGGTCGGCACGTTT）引物IGS的扩增，以及Taq DNA聚合酶的14 10 U。 </li><li>进行PCR反应使用标准的PCR参数为IGS放大：95°C / 5分钟×1周期（95°C / 30秒，50°C / 30秒，72°C / 30秒）×30个循环; 72°C / 5分钟1个循环;和4℃保持14。 </li><li>在-20℃下，于黑暗处专区探针。 </li></ol> 4。 荧光原位杂交<eM&gt;此的FISH协议包括两个变化：第一，使用BAC克隆DNA为探针的有丝分裂染色体的AE。埃及伊蚊和CX。致倦库蚊和使用IGS rDNA在有丝分裂染色体的一个第二。冈比亚。如果使用BAC克隆DNA探针，跳过核糖核酸酶处理步骤4.3，4.4，同时幻灯片/探针变性步骤4.19。如果使用的是IGS rDNA探针，杂交液没有准备C 0 T-DNA部分，并跳过单独的幻灯片/探针变性步骤，4.10，4.11，4.16和4.17。 <ol><li>孵育30分钟的幻灯片中的2×SSC在37°C。 </li><li>幻灯片系列的70％，80％和100％的乙醇脱水为5分钟，在室温下，空气干燥。 如果BAC克隆DNA进行FISH，直接进入步骤4.5。 </li><li>在0.1毫克/毫升RNA酶下石蜡溶液在37℃下的30分钟的孵育制备染色体。 </li><li>的2×SSC中洗两次，每次5分钟，在37℃下</li><li>把幻灯片我NA罐0.01％的胃蛋白酶和0.037％的HCl溶液，孵育5分钟，37°C。 </li><li> 1X PBS清洗幻灯片，在室温下5分钟。 </li><li>用1％福尔马林在1x PBS制备10％中性缓冲福尔马林浸泡10分钟，在室温下制备染色体，固定在一个罐子里。 </li><li> 1X PBS清洗幻灯片，在室温下5分钟。 </li><li>幻灯片系列的70％，80％和100％的乙醇脱水为5分钟，在室温下，空气干燥制剂，在37°C. 如果执行FISH与IGS，请直接进行步骤4.12 </li><li>变性幻灯片在一个罐子里，在72℃预热70％甲酰胺，2分钟</li><li>串联的冷（-20℃）的70％，80％，和100％乙醇，每次5分钟，并空气干燥，在37℃下脱水的幻灯片</li><li> 对于与IGS的FISH准备杂交混合物：从第3步中，10微升的C0 吨从步骤2与最终浓度为0.5毫微克/微升，和5μl1微克/微升的超声处理的鲑鱼精子DNA的DNA的标记的DNA探针的5微升。 rDNA序列，准备杂交混合物，没有C 0 T-DNA的分数。 </li><li>通过加入0.1体积的3M醋酸钠和2倍体积的乙醇​​沉淀DNA。保持在-20℃下1-3小时。 </li><li>离心机在14,000 rpm下于4℃下20分钟，除去乙醇，并空气干燥沉淀在RT。 </li><li>彻底溶解沉淀，将10μl杂交缓冲液：50％甲酰胺，20％硫酸葡聚糖，2×SSC。 如果执行与IGS FISH，直接前进到步骤4.18 </li><li>变性杂交混合物7分钟，在97℃下，并立即置于冰上1分钟。 </li><li> Prehybridize混合物在37℃下30分钟，以防止非特异的重复DNA杂交的染色体。 </li><li>将10μL杂交液在幻灯片上，和一个22X22盖玻片盖。防止泡沫的形成-温和的压力盖玻片，气泡应该被删除。 如果要进行FISH，BAC克隆DNA，直接进行步骤4.20 &lt;/ em&gt;的</li><li>变性5分钟，同时使用在75℃的加热块的探针与染色体DNA。 </li><li>在周边用橡皮泥胶盖片。 </li><li>在潮湿的室内进行杂交过夜，在37°C </li><li>从幻灯片中删除橡皮泥和盖玻片。 </li><li>洗净幻灯片2分钟，在73℃预热的解决方案1（0.4倍SSC，0.3％NONIDET-P40） </li><li>洗幻灯片的解决方案2（2×SSC，0.1％NONIDET-P40），在室温下5分钟。 </li><li>染液在1x PBS，使用0.001毫YOYO-1为10分钟，在RT下在潮湿的腔室的滑动。 </li><li>安装在少量延长Gold抗淬灭试剂与盖玻片。 </li><li>在荧光显微镜下以1,000×放大倍数（ 图4）中使用适当的过滤器组制剂分析。 </li></ol> 5。代表性的成果昆虫的ID是位于在各分部幼虫。根据不同的位置上，第EY转换成不同的组织，在成年阶段的昆虫。的ID，用于在本协议中的染色体制备，发展成在成人阶段的蚊子腿。这些ID是位于腹侧幼虫胸部和通过角质层，在显微镜下（ 图1）是清晰可见的。在早期的4 龄幼虫期，利有一个圆形的形状（ 图1A）。数目最多的有丝分裂，〜175 9，在一个ID被累积在较后的“椭圆形”的阶段（ 图1B），它必须被认为是最佳的为幻灯片准备阶段。此时，中间的ID分裂成两个：1变换到腿并和另一个变换成一翼。在“椭圆形”的阶段，我们更倾向于使用大腿部的ID的染色体玻片制备的。 图1C代表的ID在第 4龄幼虫发展的最新阶段。在这个阶段，在标识已经发展到腿和翅膀，并包含一个显着的分化组织和数量较少的有丝分裂。此ID的发展阶段，应避免染色体玻片制备。我们还建议饲养蚊子的幼虫在低温：16°C为按蚊，白纹伊蚊和致倦库蚊和22°C，这有助于增加量的有丝分裂的ID 9。 图2显示了ID解剖，从4 龄幼虫的胸部。由于角质层的一个活的昆虫是很难剖析，我们建议您使用解剖剪刀，针，而不是常用的幼虫准备。最关键的是获得高品质的染色体制备过程在低渗溶液处理。为了达到最佳效果，我们消除了肠和脂肪体从幼虫胸部在此之前治疗。在此过程中的ID细胞肿胀，有助于分散染色体上一张幻灯片（ 图3A）。低渗溶液处理的适当质量的，可以容易地识别由圆形状的细胞的准备（ 图3A，B）。一个椭圆形的形状的细胞表明不足低渗溶液处理（ 图3C）。选择鱼，制备染色体，应该包含至少50个高品质的染色体利差。通常，用于FISH 9〜90％使用该协议制备幻灯片有足够的质量。 我们提出两个稍微不同的的FISH协议：鱼基因组BAC克隆DNA探针上的白纹伊蚊和致倦库蚊的有丝分裂染色体和一个简单的协议IGS rDNA探针按蚊的有丝分裂染色体上FISH先进的协议。 白纹伊蚊和致倦库蚊的基因组是高度重复的，因为人数过多的转座因子7,8。因此，进行鱼，UTilizes基因组的BAC克隆DNA作为探针，需要加入未标记的重复DNA组分的探针，以阻止染色体的DNA重复序列的非特异的杂交。如果在重复的DNA组分的提取中，基因组DNA变性，在120℃持续2分钟。沸DNA在一个较高的温度下也有利于在200-500 bp的片段，得到DNA。 DNA被允许这种处理后，重新关联。高度重复的DNA片段往往会发现他们的队友重新组合的速度比DNA具有独特的序列。其结果是，重连接的DNA如下的 C 0×t曲线， 其中 C 0是单链DNA的初始浓度， 和 t是再退火时间。 C 0，t值等于10-4 - 10-1 100-102被认为是DNA部分中度和高度重复。不同的C 0 T-DNA组分的重新组合的时间可以计算日Ë 公式 T = C 0 T X×4.98 / C，T -时间的潜伏期，C0 吨 X - Ç0 吨分数（C0T 1 = 1，C T 2 = 2等）和C 0 -最初的15微克/微升的DNA浓度（表1）。重连接后，使用S1核酸酶消化单链DNA。我们更喜欢使用所有的C0 吨的 DNA部分C 0 T 3，而不是常用的C 0 T 1 DNA片段。 这些 C0 吨部分的包括一些中度重复DNA序列，并连同通常的35-50％的原量的基因组DNA在AE。埃及伊蚊 。标记的DNA探针和未标记的C 0 T-DNA分数之间的正确比例的重复DNA依赖于每一个特定的BAC克隆组成部分。平均而言，我们1:20探测到 C0 吨为获得一个可以接受的信号/背景比，FISH结果的DNA部分的比例。 与 C0 吨 DNA组分在管中的30分钟之前，实际在幻灯片上杂交的DNA探针的预杂交还有助于降低背景。标记，杂交，并在该协议中进行洗涤，使用标准的条件12。 两个不同的标记BAC克隆DNA探针的AE的有丝分裂染色体上的FISH结果。埃及伊蚊和Cx的。家蚊示于图4A和B，分别。 BAC克隆DNA探针在一个单一的染色体上的位置产生强烈的信号。在图1中所示的染色体YOYO-1碘化物复染。这种染料生产的最好的带型AE。埃及斑蚊染色体9。另外，其他的荧光染料，如DAPI或碘化丙啶，可用于第E染色体counterstaining。我们为了抑制光漂白的幻灯片，使用延长Gold抗淬灭安装介质。此试剂具有良好的信号保存能力，并且也可以很容易地从滑动通过在1x PBS漂洗，如果它是必要的，使用相同的几个杂交滑动除去。 一个简单的版本是专为IGS rDNA探针杂交按蚊的有丝分裂染色体上的FISH协议。 按蚊核糖体基因被表示为一个多态簇的基因位于性染色体16。在该协议中被标记的DNA探针，使用标准的PCR反应加入荧光标记Cy3或Cy5的dNTPs。因为阻断无需重复DNA在常染色质的非特异性杂交的，所有的步骤有关使用 C0 吨 DNA馏分的删去。相反，染色体准备用核糖核酸酶进行预处理，以防止IGS rDNA探针杂交核仁。染色体和DNA探针变性，同时通过加热在幻灯片杂交系统中的探针一起在75℃下5分钟。在该协议中的杂交和洗涤也使用标准的条件进行FISH 12。 FISH结果表明在图4C：IGS rDNA序列之间的两条X染色体杂交的遗传多态性是清晰可见的。 <table border="1"><tbody><tr><td fo:width="1in"></td><td fo:width="1in"> DNA浓度μg/μL </td><td fo:width="1in"> 再退火时间，min </td></tr><tr><td rowspan="6"> C 0 T 2 </td><td> 0.1 </td><td> 100 </td></tr><tr><td> 0.3 </td><td> 33 </td></tr><tr><td> 0.5 </td><td> 20 </td></tr><tr><td> 0.7 </td><td> 14 </td></tr><tr><td> 0.9 </td><td> 11 </td></tr><tr><td> 1 </td><td> 10 </td></tr><tr><td跨行=“6”&gt; C T 3 </td><td> 0.1 </td><td> 150 </td></tr><tr><td> 0.3 </td><td> 50 </td></tr><tr><td> 0.5 </td><td> 30 </td></tr><tr><td> 0.7 </td><td> 21 </td></tr><tr><td> 0.9 </td><td> 17 </td></tr><tr><td> 1 </td><td> 15 </td></tr></tbody></table> 表1中。DNA浓度和用于制备的C 0 C 0 t2和t3的馏分再退火时间。 <img alt="图1" src="/files/ftp_upload/4215/4215fig1.jpg" /> 图1，在第 4龄幼虫的ID发展阶段：A）一种早期的“圆形”的阶段，B）的中间的“椭圆形”的阶段-最优的染色体制剂; C）一种后期-染色体制剂不合适内容。幼虫胸部上的腹侧的箭头表示的位置的ID。 <img alt="图2" src="/files/ftp_upload/4215/4215fig2.jpg" /> 图2。步骤编号 ​​夹层：甲）断头幼虫（切口的方向被表示由箭头）; B）的幼虫与解剖肠道低渗溶液处理下（ID的溶胀，变得几乎不可见）;Ç）幼虫卡诺溶液应用程序后（ ID变成白色的和清晰可见的），D）解剖卡诺溶液中的ID。在幼虫的ID位置的星号表示。 <img alt="图3" src="/files/ftp_upload/4215/4215fig3.jpg" /> 图3不同品质的染色体差：A）一种完美的染色体蔓延-圆形的细胞表现出足够的治疗在低渗溶液中的ID; B）一种完美的低渗处理-染色体稍微undersquashed; C）一种染色体蔓延差 - 的结果低渗处理不足指示由椭圆形的细胞。 <img alt="图4" src="/files/ftp_upload/4215/4215fig4.jpg" /> 图4。鱼BAC克隆（A，B）和IGS rDNA序列在染色体上的AE（C）的例子。埃及斑蚊（A），CX致倦库蚊（B），以及。冈比亚按蚊 （C）。 1，2和3 - ，X -在一种女性性染色体的染色体数目。冈比亚。

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没有利益冲突的声明。

非荧光原位杂交技术在有丝分裂染色体的蚊子进行了第一次在1990年由A. Kumar和K.清莱17。在这项研究中，18S和28S核糖体DNA基因，克隆在一个质粒一起，被安置到染色体的20个种类的蚊子。放射性标记的DNA探针和杂交的染色体大脑的神经。在三个蚊属，FISH技术已发展有丝分裂染色体的细胞系AE。埃及伊蚊 10,18,19和从未有丝分裂染色体进行现场蚊子。最近，我们开发了一个简单，可靠的技术，获得高品质的染色体准备从第 4龄幼虫的ID。此方法允许在一个幻灯片方式获得高的染色体数目，可以普遍适用于所有种类的蚊子。只幼虫，蛹或成人站的必要性GES蚊子，玻片制备的方法可能是唯一的限制。标准FISH方法进行了优化，利用基因组BAC克隆和IGS rDNA序列作为探针， 白纹伊蚊，库蚊，按蚊的有丝分裂染色体的。 除了这些特定的应用程序，这里描述的FISH协议也可以被用于其他目的。先进的的FISH协议，利用C 0 T-DNA用于阻断非特异性杂交的馏分，也可以应用于用于杂交的BAC克隆或任何其他的大的DNA片段在按蚊异染色质区域。异染色质区域丰富的转座因子和其他的重复，而来自这些地区的探针产生强烈的背景染色体上的3。使用未标记的C 0 T-DNA部分，将有助于减少非特异的探针杂交的染色体。简单版本的鱼PRotocol可用于任何rDNA或有丝分裂染色体的重复DNA探针上的蚊子和其他昆虫。此外，它也可以适用于BAC克隆DNA的杂交物种按蚊或果蝇常染色质区域，如具有低重复DNA含量。这里提出的协议将有助于获得高成品染色体的基因组的蚊子，可广泛用于各种细胞遗传学的应用程序在其他组的昆虫。

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> MZ6徕卡体视显微镜</td><td>徕卡</td><td> VA-OM-E194-354 </td><td>可以使用一个不同的体视显微镜</td></tr><tr><td>奥林巴斯CX41相差显微镜</td><td>奥林巴斯</td><td> CX41 </td><td>不同的相显微镜可用于</td></tr><tr><td>奥林巴斯BX61荧光显微镜</td><td>奥林巴斯</td><td> BX61 </td><td>可用于不同的荧光显微镜</td></tr><tr><td> ThermoBrite幻灯片变性/杂交系统</td><td>雅培分子</td><td> 30-144110 </td><td>作为一个加热块和潮湿的腔室</td></tr><tr><td>解剖针</td><td>精细ScienceTools </td><td> 10130-10</td><td></td></tr><tr><td>持针器</td><td>精细科学工具</td><td> 26018-17 </td><td></td></tr><tr><td>解剖剪</td><td>精细科学工具</td><td> 15000-03 </td><td></td></tr><tr><td> 75x25双磨砂微滑动</td><td>康宁</td><td> 2949-75x25 </td><td></td></tr><tr><td> 22x22毫米显微镜盖玻片</td><td> Fisher Scientific则</td><td> 12-544-10 </td><td></td></tr><tr><td>封口膜</td><td> Fisher Scientific则</td><td> 13-374-10 </td><td></td></tr><tr><td>橡皮泥</td><td> Fisher Scientific则</td><td> 50-949-105 </td><td></td></tr><tr><td>醋酸</td><td> Fisher Scientific则</td><td> A491-212 </td><td></td></tr><tr><td>酒精200的证明</td><td> DECON实验室</td><td> 2701 </td><td></td></tr><tr><td>丙酸</td><td> Sigma-ALDR的非物质文化遗产</td><td> 402907 </td><td></td></tr><tr><td>盐酸</td><td> Fisher Scientific则</td><td> A144-500 </td><td></td></tr><tr><td>柠檬酸钠二水合物</td><td> Fisher Scientific则</td><td> S279-500 </td><td></td></tr><tr><td>三水醋酸钠</td><td> Fisher Scientific则</td><td> BP334-500 </td><td></td></tr><tr><td>氯化钾</td><td> Fisher Scientific则</td><td> BP366-500 </td><td></td></tr><tr><td> EDTA </td><td> Fisher Scientific则</td><td> S311-500 </td><td></td></tr><tr><td>三基地</td><td> Fisher Scientific则</td><td> BP152-1 </td><td></td></tr><tr><td> 10倍PBS </td><td> Invitrogen公司</td><td> P5493 </td><td></td></tr><tr><td> 10％NBF（中性福尔马林缓冲液） </td><td> Sigma-Aldrich公司</td><td> HT501128 </td><td></td></tr><tr><td> 99％甲酰胺</td><td>费舍尔SCIE科学研究， </td><td> BP227500 </td><td></td></tr><tr><td>硫酸葡聚糖钠盐</td><td> Sigma-Aldrich公司</td><td> D8906 </td><td></td></tr><tr><td> 20X SSC缓冲</td><td> Invitrogen公司</td><td> AM9765 </td><td></td></tr><tr><td> 1毫米YOYO-1碘（491/509）解决方案</td><td> Invitrogen公司</td><td> Y3601 </td><td></td></tr><tr><td>抗淬灭延长黄金试剂</td><td> Invitrogen公司</td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=P36930" target="_blank">P36930</a> </td><td></td></tr><tr><td>的dATP，dCTP，dTTP的，三磷酸</td><td> Fermentas公司</td><td> R0141，R0151，R0161，R0171 </td><td></td></tr><tr><td>的Cy3，Cy5-dUTP标记的dUTP </td><td> GE医疗集团</td><td> PA53022，PA55022 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich公司</td><td> A3294 </td><td></td></tr><tr><td> DNA聚合酶I </td><td> Fermentas公司</td><td> EP0041 </td><td></td></tr><tr><td> DNA酶I </td><td> Fermentas公司</td><td> EN0521 </td><td></td></tr><tr><td> S1核酸酶</td><td> Fermentas公司</td><td> EN0321 </td><td></td></tr><tr><td> Taq DNA聚合酶</td><td> Invitrogen公司</td><td> 18038-042 </td><td></td></tr><tr><td>核糖核酸酶</td><td> Sigma-Aldrich公司</td><td> 9001-99-4 </td><td></td></tr><tr><td>胃蛋白酶</td><td> USB </td><td> 9001-75-6 </td><td></td></tr><tr><td>鲑鱼精子DNA </td><td> Sigma-Aldrich公司</td><td> D7656 </td><td></td></tr><tr><td> NONIDET-P40（NP40） </td><td>美国生物</td><td> NC9375914 </td><td></td></tr><tr><td> Qiagen公司的血液和细胞培养Maxikit </td><td> Qiagen公司</td><td> 13362 </td><td></td></tr><tr><td> Qiagen公司的大构造工具包</td><td> Qiagen公司</td><td> 12462 </tD&gt; <td></td></tr></tbody></table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

荧光原位杂交（FISH）是一种技术，经常使用的许多实验室，以确定染色体的DNA和RNA探针的位置。这种方法的一个重要的应用是有用的，用于改善各种生物体的基因组组件的高品质的物理图谱的发展。自然的的唾腺和有丝分裂染色体带型的基因组supercontigs精确的顺序和方向提供指导。在这三个蚊属， 按蚊 ， 伊蚊，库蚊 ，一个完善的染色体为基础的绘图技术已发展按蚊 ，其成员均具备可读的多线染色体1。作为基因组作图的努力的结果，88％的一种。冈比亚按蚊的基因组已被放置到准确的染色体位置2,3。另外两个蚊属， 伊蚊和库蚊，不善，polytenized染色体，because的显着比例过高的转座因子的基因组4，5，6。无秩序或定位到染色体AE只有31和9％的基因组supercontings已被分配。埃及伊蚊7和CX。致倦库蚊 。这两个物种的细胞有丝分裂染色体制备了以前仅限于大脑的神经和细胞系。然而，染色体来自大脑的神经节的蚊子准备的幻灯片通常含有低的中期板9。此外，虽然FISH技术已发展为一个细胞的有丝分裂染色体的AE。埃及斑蚊 10，细胞染色体的11多个染色体重排的积累，使他们无用的基因组图谱。在这里，我们描述了一个简单，强大的技术获得高品质的有丝分裂染色体的准备，从4 龄幼虫的成虫盘（IDS）的其中c可用于所有三个属的蚊子。优化使用BAC克隆的基因组DNA为探针的有丝分裂染色体的AE一个标准的FISH协议12。埃及伊蚊和Cx。家蚊，和利用的间隔区（IGS）的核糖体DNA（rDNA）的区域上的一个作为探针。冈比亚按蚊染色体。除了到物理地址的映射，开发的技术可以应用到人口的细胞遗传学和染色体分类/蚊子和其他昆虫系统学的。

Watch this Scientific Journal Video about Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes at JoVE.com

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

其中三个蚊属，即<em&gt;按蚊，</em&gt;<em&gt;白纹伊蚊</em&gt;，和<em&gt;致倦库蚊</em，身体的基因组图谱技术建立了仅适用于<em&gt;按蚊</em&gt;，其成员具有可读的多线染色体。对于属<em&gt;白纹伊蚊</em&gt;和<em&gt;致倦库蚊</em，但是，细胞遗传学映射仍然是具有挑战性的，因为质量差的多线染色体。在这里，我们提出了一种通用协议获得高品质的有丝分裂染色体的准备和优化的FISH协议的所有三个属的蚊子。

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

fluorescent-in-situ-hybridization-on-mitotic-chromosomes-of-mosquitoes

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JoVE Journal

Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

25.5K 次观看.  Virginia Tech. 其中三个蚊属，即按蚊，白纹伊蚊，和致倦库蚊

视频: Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

疟疾蚊子中的二维和三维染色体绘画

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

在伊蚊中传播微孢子寄生虫Edhazardia aedis

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...