我们感谢N.院长提供原mCherry FP序列，M Gerami - 内贾德建设质粒，拉尔森B.技术援助和T.海泽尔这个项目的开发过程中有益的建议。 JB是由欧洲研究委员会高级奖340087（RAPLODAPT）的支持。显微及成像系统是由明尼苏达州儿科基金会大学和明尼苏达影像中心的大学提供。

1.切断模板质粒从大肠杆菌 <ol><li>生长含模板质粒过夜在10ml LB培养基（LB）+ 200毫克/升氨苄青霉素（AMP），在37℃振荡的大肠杆菌 。 </li><li>收获细胞通过在6000 xg离心离心2分钟。 </li><li>倾析液体，分离和纯化的DNA从大肠杆菌如Ausubel 等人以前描述通过标准方法的大肠杆菌细胞。 8。 </li><li>在50-100 ng / ml的工作浓度;重悬的DNA在Tris-EDTA（10毫摩尔Tris，pH值8.0，1mM EDTA中，pH值8.0，TE）。 </li></ol> 2.设计引物<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td fo:width="0.5in" style="width:50px"> 底漆 </td><td> 引物序列 </td></tr><tr><td> F1 </td><td> 5&#39; <strong&gt; GAGAATTGAAGAAGAGTTGGGAGACAATGCTATCTATGCTGGTAAGGACTTCCACAATGCTCAAACTTTG GGTGGTTCTAAAGGTGAAGAATTATT 3&#39; </td></tr><tr><td> R1 </td><td> 5&#39;GAGCGTTTGCACCAACAGGCCATCATTTGTGACGAGAGAAGACCTGACGTCATTAGATTGGCACCTTTGC GTAAAACGACGGCCAGTGAATTC 3&#39; </td></tr><tr><td> F2 </td><td> 5&#39;GAGAATTGAAGAAGAGTTGGGAGACAATGCTATCTATGCTGGTAAGGACTTCCACAATGCTCAAACTTTG GGTGGTGTTTCAAAAGGTGAAGAAGATAAT 3&#39; </td></tr><tr><td> R2 </td><td> 5&#39;GAGCGTTTGCACCAACAGGCCATCATTTGTGACGAGAGAAGACCTGACGTCATTAGATTGGCACCTTTGC ACTGGATGGCGGCGTTAGTATC 3&#39; </td></tr></tbody></table> 表1:在本研究中使用的引物序列。粗体斜体文字表示同源性的ENO1基因组基因座，通常字体区是同源的质粒DNA。 <ol> &lt;利&gt;设计的引物是同源质粒接壤的带盒的序列被扩增以及所关注的靶基因（ 例如 ，ENO1）的3&#39;端，以促进重组到基因的基因组基因座（ 图2和表1） 。 <ol><li>保证正向引物的序列相匹配的最后70个碱基对的目的基因的（BP），5&#39;-3&#39;，减去终止密码子，以保持编码帧，以及所述第一约30碱基的质粒序列的要放大。作为一个说明，在各引物的GGTGGTGGTT是聚 - 甘氨酸接头没有FP同源性。值得注意的是，在不存在连接体的，可以有功能结构域，其可以理论上导致蛋白质错误折叠，低收率在蛋白质生产，或者受损的生物活性的直接融合。 </li><li>确保反向引物的序列是70碱基只是基因的下游，3&#39;- 5&#39;，不包括任何基因序列，加上最后的Approximately在质粒中使用的营养或药物抗性标记的30碱基对。 </li><li>使用F1和R1产生GFP- NAT1和YFP- NAT1盒，带pMG2120和pMG2263，分别F2和R2产生mCherry- NAT1与pMG2343。 </li></ol></li></ol> 3.通过PCR生成FP盒（1日） <ol><li>准备试剂PCR。使预混液（500微升最终体积）加入下面的体积和浓度为1.5 ml管:50微升PCR缓冲液（500毫米氯化钾，100毫米的Tris pH值8.0，在水中），20微升脱氧核苷酸（的dNTP;股票混合在每10毫摩尔），40微升的25mM氯化镁核苷酸，20微升纯化的质粒（从〜50-100纳克/微升原液）中，每个前向和反向引物（从10mM储备溶液10微升），30微升的Taq聚合酶（通用，5000单位/ ml），和320微升水。 <ol><li>分装50μl反应混合液到每10 PCR-compatib的乐0.5毫升管。 </li></ol></li><li>将PCR管在热循环仪和执行下列步骤:周期分5 1 94℃变性双链DNA;顺序地45秒的40个循环的94℃，在55℃30秒，以允许引物退火到质粒模板DNA，并在68℃的DNA产物延伸4分钟;并在72℃下的15分钟1最终延伸循环。 注:PCR主混合物和循环参数可能需要根据所使用的特定的Taq聚合酶进行修改。 </li><li>池从1.5毫升管的10个PCR反应的所有产品。 <ol><li>主体5微升汇集的PCR产物的琼脂糖凝胶电泳来验证扩增片段的大小，将获得产物浓度的估计值，基于比较的DNA梯。一般情况下，使用〜250微克盒式DNA在以后的每个转化混合物。 </li><li>通过加入乙酸将50μl的3M钠后跟750微升95％乙醇的产物沉淀DNA并孵育至少30分钟在-20℃。 </li><li>由16,000 XG离心管10分钟收获PCR产物。小心取出并弃去上清液和隔夜干燥沉淀。重悬在40微升TE pH为8.0的干燥的DNA盒沉淀并在室温下储存，直到使用。 </li></ol></li></ol> 4. 念珠菌转化细胞的DNA FP磁带<ol><li>在第1天，回收酵母菌株以从15％甘油冷冻（-80℃）的库存划线几刮取结晶到酵母蛋白胨葡萄糖腺嘌呤（YPAD）琼脂并于30℃培养被转化。菌落生长的恢复后，接种单个菌落于2ml液体YPAD培养基在具有透气盖的玻璃培养管中并在30℃搅拌孵育过夜。 </li><li>在第2天，在125毫升锥形瓶中用透气帽稀释300微升过夜酵母培养物于50ml新鲜YPAD（至最终OD 600〜0.2）。摇在30℃对于〜3小时（到最终OD 600〜0.6-0.8）。 <ol><li>倾过夜培养到50毫升锥形管，并通过在台式离心机在1 500 xg离心纺丝5分钟沉淀细胞。 </li><li>倒出并正确丢弃上清液。重悬在5ml水中的细胞沉淀。通过在1,500 XG在台式离心机再次离心5分钟重新沉淀细胞。 </li><li>倒出并正确丢弃上清液。悬浮于500μlTELiAc（TE乙酸锂:10毫摩尔Tris，pH值8.0，1mM EDTA中，pH值8.0，0.1M乙酸锂）的细胞，而转移到1.5ml管中。离心管2分钟在一个离心3000 XG。 </li><li>悬浮细胞在250微升TELiAc。总体积包括颗粒应为〜300微升。 </li></ol></li><li>到一个干净的（不同的离心管比4.2.4），加5微升载体DNA（10毫克/毫升），并准备念珠菌细胞的150微升（从4.2.4）。这是在t阴性对照ransformation。 <ol><li>到第二清洁离心管中，添加5微升变性载体DNA（已在90℃煮沸10分钟，并冷却至4℃），将制备的PCR产物的所有40微升（从3.3.1）和150准备念珠菌细胞微升（从4.2.4）。 </li><li>孵育在室温下30分钟两变换混合。 </li><li>给每个转化混合物管中，加入700μl板混合物（10毫摩尔Tris，pH值8.0，1mM EDTA中，pH值8.0，0.1M乙酸锂在50％聚乙二醇3350）。颠倒管混匀，在室温孵育过夜他们。 </li></ol></li><li>在第3天，孵育转化在42℃1小时（热休克）混合。 <ol><li>离心机改造30秒在微量16000 XG混合。取出并妥善弃去上清液。轻轻吹打向上悬浮每150微升水中的细胞沉淀的上下，从而不会损坏电池。 </li><li>对于UT变换ilizing营养缺陷型标记基因（ 例如 URA3），吹打解决方案到适当的选择性培养基琼脂（ 如缺乏尿苷）板每整个混合物均匀涂抹，用无菌玻璃珠混合物。 </li><li>用于利用诺尔丝菌素抗性标记基因（NAT1）转换，如这里描述的，板转换第一混合到非选择性YPAD琼脂并于30℃下孵育6-12小时。这一步骤有助于在细胞恢复，后期热休克，应激诺尔丝菌素之前被应用。 </li><li>后部分恢复性增长，副本板念珠菌细胞上含有YPAD 400微克/毫升诺尔丝菌素。对于利用的营养标记基因（ 例如 URA3）变换，则不需要此中间镀敷工序和细胞可以直接镀到如在4.4.2中描述的选择性酵母媒介（ 例如 YPAD缺乏尿苷）。 注:如果改造是成功的，山坳onies应在一到三天出现（可能高达生长的五天选择含诺尔丝菌素琼脂）。没有殖民地应该出现在含载体DNA本身（阴性对照）改造混合传播板。 </li></ol></li><li>为营养缺陷型和诺尔丝菌素标记选择，条纹推定的转化单菌落至新鲜选择培养基琼脂平板上，孵育在30℃下传播，可筛选的FP融合的成功构建酵母细胞。 </li><li>屏幕为转化整合正确标注该盒（见一个详细的例子代表性的成果 ）。如果感兴趣的基因在足够量的表达时，可能会发生这样的整个群体的荧光，这是可能的，以检测使用荧光检测能力的印版成像系统的潜在候选整合体。 <ol><li>通过使用PCR引物同源序列outsid检查假定的整合一体化的区域e确认融合到靶基因。 </li><li>此外，考虑Western印迹分析，以确定融合蛋白的表达和大小，以及由单细胞蛋白质定位的视觉确认，如果已知的荧光显微镜。 </li></ol></li></ol>

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作者什么都没有透露。

表位的结构标记在使用上述的PCR介导的基因修饰策略念珠菌物种的序列可以归纳为一个三步过程。首先，盒通过PCR编码既期望集成和区域同源的插入轨迹入酵母基因组中的序列进行。第二，要转化的酵母细胞制成用乙酸锂和共同培养与盒化学感受。第三，将细胞铺于选择性培养基以回收转化体和所得菌落为正确整合盒成所需基因组基因座的测试。 有此协议内的几个关?...

念珠菌是殖民所有人类的肠道和泌尿生殖道共生真菌。下免疫缺陷的情况下，如发生与早产或从癌症治疗的免疫抑制作用， 念珠菌物种可以成为机会致病菌。 念珠菌物种的， 白色念珠菌是最普遍的真菌殖民者并且使得大多数侵袭性真菌感染。其它念珠菌属种类，如光滑念珠菌 ， 近平滑念珠菌 ， 热带念珠菌和C. kruseii也造成免疫功能低下患者的严重感染，有些显示出固有电阻对常用抗真菌抗生素如氟康唑和两性霉素B因此，与这些物种的感染正在观察更频繁，特别是在被用抗真菌剂预防性治疗的患者。即使有适当和及时的一NTI真菌治疗，侵袭性念珠菌感染继续与显著的发病率和死亡率的1个相关。因为在人体健康念珠菌属种类的意义，有必要为现成的分子工具，允许它们的发病机理的研究和澄清。 其中一个重要的工具，它使研究人员能够可视化和量化微生物细胞，他们表达的蛋白质是FP融合技术。聚合酶链反应（PCR）介导的基因修饰，如在本文中所描述，使融合的构建，FP序列和念珠菌蛋白在其基因组基因座编码的感兴趣序列之间。构建体的稳定整合促进蛋白表达的分析以及蛋白质定位动力学。含有FP序列的质粒，在白色念珠菌中表达优化的，所用的PCR介导的用于g烯改性的策略，已经预先构造2，3，4，5。质粒含有FP转换"盒":链接到有利于白色念珠菌和近平滑念珠菌 2，3，4，5，6，7的转化的营养标记基因的FP序列。目前可获得的质粒含有多种对营养缺陷型菌株的转化选择性营养标记基因（URA3，HIS1，ARG4）以及显性药物抗性标记（NAT1），这有利于缺乏营养缺陷型临床株的转化。此外，质粒含有多达四个不同的FP序列（绿色[GFP]选项黄釉W [YFP]，青色[CFP]和樱桃[mCherry]）和任一用于施工羧基末端蛋白融合物，或用于建筑氨基末端蛋白质融合的启动子序列的ADH1终止序列。引物设计有同源性的周围在FP带盒的质粒DNA。此外，引物还包含轴承的同源性的感兴趣的酵母基因进行标记，这有利于带盒的整合到通过同源重组（ 图1）的基因组基因座5&#39;延伸序列。基因特异性FP盒通过PCR生成，然后转化到通过用乙酸锂处理而处于感受态用于DNA的摄取念珠菌细胞。 <img alt="图1" src="/files/ftp_upload/55333/55333fig1.jpg" /> 图1:FP序列融合如何在念珠菌属种类产生的图。 （A）质粒DNA大型的源码ES一个FP序列和编码序列诺尔丝菌素抗性（NAT1）。正向（FWD）和反向（REV）的引物的相对位置示出，以指示同源的区域与质粒序列的引物和表示特定基因同源区或引物延伸的紫色部分的黑色部分。 （B）中的FP盒被变换成念珠菌和通过同源重组（虚线）的ENO1基因座内集成。 （C）在ENO1的3&#39;末端所得的FP融合序列。 <a href="http://ecsource.jove.com/files/ftp_upload/55333/55333fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在此，我们提出在念珠菌蛋白融合物（左心室Eno1-FP）的结构的一个例子。我们使用含标记与编码GFP，YFP序列沿着NAT1转化标记基因的质粒，或mCherry（ 图2）。将这些质粒与在PCR引物一起使用，以产生特定基因盒便于FP的融合ENO1的3&#39;末端，从而导致在其羧基末端融合到的FP左心室Eno1的表达。 <img alt="图2" src="/files/ftp_upload/55333/55333fig2.jpg" /> 图2:FP含盒式质粒的图谱。向前（F）和用于产生从所述质粒卡匣倒车档（R）的引物与它们的同源性的质粒的相对位置沿指示。引物序列如表1所列。 F1和R1也被用来生成所述pYFP- NAT1盒。含有YFP- NAT1盒（pMG2263）质粒是与YFP的代替GFP序列的异常相同pMG2120。盒尺寸:GFP-NAT1，3.7 KBP; mCherry- NAT1，3.2 KBP; YFP- NAT1，3。7 KBP。这个数字已经从Gerami -内贾德， 等修改。 4 <a href="http://ecsource.jove.com/files/ftp_upload/55333/55333fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>100 W mercury lamp</td><td>CHIU Technical Corporation</td><td>M-100T</td><td></td></tr><tr><td>95% Ethanol</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Adenine</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Ampicillin</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Carrier DNA</td><td>Ambion</td><td>AM9680</td><td>Sheared Salmon Sperm DNA 10 mg/ml</td></tr><tr><td>CCD Camera</td><td>Photometrics</td><td>CoolSNAP HQ</td><td></td></tr><tr><td>Conical Tube</td><td>Corning</td><td>430828</td><td>50 ml</td></tr><tr><td>Culture Tube Rotator</td><td>New Brunswick</td><td>2013923</td><td>TC-8, or Any Culture Tube Rotator</td></tr><tr><td>Deoxynucleotides (dNTP) PCR Grade</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Eppendorf Tubes</td><td>Eppendorf</td><td>022363719, 022363212</td><td>0.5 ml, 1.5 ml</td></tr><tr><td>Erlenmeyer Flask</td><td>Fisher Scientific</td><td>7250089</td><td>125 ml</td></tr><tr><td>Ethylenediaminetetraacetic Acid (EDTA)</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td>Thermo Electron Corporation</td><td>ULT-1386-9-V</td><td>Revco Ultima II</td></tr><tr><td>GFP, YFP and Texas Red Filter Sets</td><td>Chroma Technology Corporation</td><td>49002, 86004v2, 49008</td><td></td></tr><tr><td>Glass culture tubes</td><td>Fisher Scientific</td><td>1496126</td><td>75 mm</td></tr><tr><td>HRP goat anti-mouse antibody</td><td>Santa Cruz Biotechnology</td><td>SC-2005</td><td></td></tr><tr><td>HRP goat anti-rabbit antibody</td><td>Santa Cruz Biotechnology</td><td>SC-2301</td><td></td></tr><tr><td>Incubator (30 &amp;deg;C)</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Lithium Acetate</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Lysogeny Broth (LB) Media</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415 D</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>E600</td><td>Nikon Eclipse E600</td></tr><tr><td>Microscope Image Analysis Software</td><td>Universal Imaging Corporation</td><td>6.3r7</td><td>MetaMorph Software Series 6.3r7</td></tr><tr><td>Mouse anti-GFP antibody</td><td>Roche</td><td>11814460001</td><td></td></tr><tr><td>Nourseothricin</td><td>Fisher Scientific</td><td>50997939</td><td></td></tr><tr><td>PCR Thermocycler</td><td>Applied Biosystems</td><td>9700</td><td>GeneAmp PCR System</td></tr><tr><td>PCR tubes</td><td>BioExpress, GeneMate</td><td>T-3035-1</td><td>0.2 ml</td></tr><tr><td>Polyethylene Glycol 3350</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Potassium Chloride</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Rabbit anti-mCherry antibody</td><td>BioVision</td><td>5993-100</td><td></td></tr><tr><td>Refrigerator (4 &amp;deg;C)</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Sodium Acetate</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>Table Top Centrifuge</td><td>Labnet</td><td>Z 400</td><td>Hermle Z 400</td></tr><tr><td>&lt;em&gt;Taq&lt;/em&gt; DNA Polymerase</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Tris)</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Vortex Mixer</td><td>Scientific Industries</td><td>SI-0236</td><td>Vortex Genie 2</td></tr><tr><td>Yeast Extract Peptone Dextrose (YPD) Media</td><td>Any</td><td>NA</td><td></td></tr></tbody></table>

generation of fluorescent protein fusions in candida species

念珠菌属种类，肠道和泌尿生殖道的常见殖民者，是多数人类侵袭性真菌感染的原因。因此，需要分子和遗传工具，以促进它们的发病机理的研究。 PCR介导的基因修饰是产生表位标记的蛋白质以促进其检测一个简单，快捷的方式。特别是，荧光蛋白（FP）融合体是功能强大的工具，使酵母细胞和蛋白质的可视化和定量分别由荧光显微镜和免疫印迹。含FP编码序列，有利于念珠菌的改造营养标记基因的质粒一起，已经为FP建设和表达念珠菌的目的产生的。在此，我们提出了在念珠菌构建FP融合的战略。包含诺尔丝菌素resista质粒NCE变换标记基因（NAT1）与绿色，黄色，或樱桃的FP（GFP，YFP，mCherry）与引物包括在聚合酶链反应（PCR）的基因特异性序列一起使用，以产生一个FP盒序列沿。这个特定基因盒具有集成到通过同源重组的相应基因座的3&#39;端的能力。成功的框内融合将FP序列插入的感兴趣的基因位点的是基因验证，然后通过显微镜和/或免疫检测方法的融合蛋白表达的分析。此外，对于高表达的蛋白质的情况下，成功的融合可以通过荧光成像技术筛选为主。

作为一个例子，我们使用上述的协议来构建GFP和mCherry融合在一个近平滑念珠菌实验室菌株ENO1。每个推定转化最初是重新划线增长。在本实施例中，由于所得到的融合蛋白中高度表达（烯醇）和FP的明亮，我们能够进行诊断性PCR之前筛选用荧光显微镜转化体（ 图3）6。 <img alt="图3" src="/files/f...

Watch this Scientific Journal Video about Generation of Fluorescent Protein Fusions in Candida Species at JoVE.com

Generation of Fluorescent Protein Fusions in Candida Species

PCR介导的基因修饰可用于产生在念珠菌属种类，这有利于可视化和酵母细胞和蛋白质的定量荧光蛋白融合体。在此，我们提出了在近平滑念珠菌构建荧光蛋白融合（左心室Eno1-FP）的战略。

在荧光蛋白融合的一代<em&gt;念珠菌</em&gt;物种

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

generation-of-fluorescent-protein-fusions-in-candida-species

Research

JoVE Journal

Genetics

Generation of Fluorescent Protein Fusions in Candida Species

10.6K 次观看.  University of Minnesota.  PCR介导的基因修饰可用于产生在念珠菌属种类，这有利于可视化和酵母细胞和蛋白质的定量荧光蛋白融合体。在此，我们提出了在近平滑念珠菌构建荧光蛋白融合（左心室Eno1-FP）的战略。 

视频: Generation of Fluorescent Protein Fusions in Candida Species

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

科研

发育生物学

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

免疫与感染

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

<meta charset="utf8"></head><body>使用荧光蛋白标记物可视化酵母细胞器

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

使用荧光蛋白标记物可视化酵母细胞器

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

生物化学

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

生物学

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

建立 白色念珠菌相关导管相关感染的小鼠模型

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

作者聚焦:使用荧光蛋白标记增强导管感染中的白色念珠菌检测 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

小鼠导管相关 白色念珠菌 感染模型

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

在荧光蛋白融合的一代

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C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

荧光标记基因的基因枪转化成机会性真菌病原

使用荧光蛋白标记物可视化酵母细胞器

使用荧光蛋白标记物可视化酵母细胞器

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

裂殖酵母的活细胞定量荧光显微镜分析

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型

小鼠导管相关白色念珠菌感染模型

小鼠导管相关白色念珠菌感染模型

小鼠导管相关白色念珠菌感染模型

在荧光蛋白融合的一代

摘要

探索更多视频

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

荧光标记基因的基因枪转化成机会性真菌病原

使用荧光蛋白标记物可视化酵母细胞器

使用荧光蛋白标记物可视化酵母细胞器

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

裂殖酵母的活细胞定量荧光显微镜分析

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

小鼠导管相关 白色念珠菌 感染模型

小鼠导管相关 白色念珠菌 感染模型

小鼠导管相关 白色念珠菌 感染模型

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型

小鼠导管相关白色念珠菌感染模型

小鼠导管相关白色念珠菌感染模型

小鼠导管相关白色念珠菌感染模型