这项工作是从国家科学基金会（断路器-0923794金融时报）的赠款支持。

1。产生子囊壳<ol><li>中心接种胡萝卜琼脂6.0厘米直径的Petri与楼肥沃的应变菜10 禾谷 （推荐的PH-1）。 </li><li>接种在室温（18-24℃）明亮的荧光灯下的菜，让菌丝生长，直到已达到外缘的培养皿（3-5天）。商用家用的日光灯做工精细，刺激子实体的形成和排放。 </li><li>轻轻取出用无菌牙签的气生菌丝。 </li><li>分发1.0毫升2.5％吐温60水溶液的表面用无菌玻璃曲棍球棒或无菌玻璃棒圆润结束。 </li><li>返回光板。不添加石蜡膜板。 </li><li> 24小时后，板表面应该有一个闪亮的外观。如果再次出现菌丝体，刮去表面和重新申请吐温60的解决方案。 </li><li>在未来一周观察的子囊发展。中间阶段子囊发展可收获轻轻刮表面冻结提取RNA（ 图1）琼脂和闪光灯。 </li><li>成熟的孢子会积聚在7天板盖。最初，这些只会在解剖显微镜下可见，但9天，他们将已经积累了足够的密度，以肉眼可见。 </li></ol> 2。 。子囊放电分析<ol><li>第6天吐温的解决方案的应用程序后，切开一个1厘米直径的圆琼脂木钎。另外，手术刀可以用来删除一个类似大小的段。圆可以被切成一半，各占一半放在玻璃显微镜幻灯片。 </li><li>东方件，所以包含子实体表面是垂直滑动面，孢子被解雇的幻灯片的长度。 </li><li>放置在湿度室的灯光下过夜的幻灯片。孢子会积聚在幻灯片和第二天早上（ 图2）肉眼可见。 </li><li>累计孢子可以洗掉的水滑梯，量化，如果需要的话。 </li><li>潜在的子囊孢子放电抑制剂可以将它们添加到琼脂块在背面装配检测检测。一些离子通道抑制剂，减少排放先前已检测用这种方法15。 </li></ol> 3。代重组后代<ol><li>发起与两楼株接种胡萝卜琼脂跨越禾谷镰刀菌 （尼特+一NIT），放置在培养皿两侧的接种点。 </li><li>一旦菌丝填补表面，刮去空中部分和申请吐温-60解决方案，如上所述。使用标记要注意培养皿菌丝的一面。 </li><li>返回光的文化和孵化，直到子囊已经制定，并cirrhi有从子囊壳沿两种文化之间的交汇，形成枪。 </li><li>解剖显微镜下，将进入查看子囊沿两种文化的交叉点。利用昆虫针，将消毒和无菌水浸泡，沿着这条边界接触1 cirrhus轻轻用针的尖端。应该让cirrhus坚持引脚引脚上的水分。 </li><li>冲洗它cirrhus在Eppendorf管洗孢子在200μL无菌水针的尖端。 </li><li>火焰脚，滋润了一遍，并选择另一个cirrhus。挑选10至15 cirrhi，并放置在每一个单独的水管。 </li><li>简要涡管含有悬浮cirrhi。 </li><li>取出60μL的孢子悬浮在9厘米的培养皿用吸管和地方MMTS介质1。用无菌曲棍球棒的传播。余下的孢子悬浮液在4°C，可存储长达一个星期，如有必要replated。 </li><li> incuba在室温为3-5天（光没有必要）TE，直到出现非常小的菌落（ 图3）。会有两种殖民地之间的表型。 NIT的殖民地，将有稀疏菌丝比尼特+克隆。从重组子囊cirrhi将显示比例大致相等的两个殖民地的表型。丢弃板，只对他们的单一型的殖民地。请注意，有时两个以上的表型隔离等因素导致因。 </li><li> V8或胡萝卜琼脂存储移动殖民地。确认正确的表型;察氏的阿霉素琼脂（NIT-株不会增长察氏的阿霉素），PDA与氯酸钾（NIT +菌株不会长0.5米PDA与氯酸钾氯酸钾）测试的殖民地。检查所需的素质这些殖民地。 </li></ol> 4。代表结果产生子囊壳我们的目标是有草坪子囊壳没有任何菌丝或孢子明显。文化的表面会出现类似的黑色天鹅绒（ 图1）。吐温的应用在24小时后，在琼脂表面有轻微的光泽。如果菌丝再现，然后板可能不会被擦到足以产生子囊发展。 子囊孢子放电始终提供您的检测琼脂的几个街区，从野生型菌株。如果这些不火，那么无论你的子囊壳是太年轻或太老。如果他们是太旧了，然后会出现大量菌丝在表面的文化给它一个白色的色调。如果不是这样的情况下，他们可能是太年轻了，该法应再次尝试在24小时。偶尔，文化不履行良好，将不得不重复实验。务必执行适当的光照条件下检测。 重组后代<img alt="图1" src="/files/ftp_upload/3895/3895fig1.jpg" /> 图1。胡萝卜琼脂子囊发展阶段。左，72小时后吐温的应用。注意红色颜料和黑色颗粒表面上可见的非常年轻的子囊壳。右，在144 h，已形成成熟的子囊壳。注意接近完成的情况下，在两种文化的肤浅菌丝。 <img alt="图2" src="/files/ftp_upload/3895/3895fig2.jpg" /> 图2。放电检测。橙色的胡萝卜琼脂插件是面向所以THA载玻片表面，ţ强行出院，从成熟的子囊壳在其表面的孢子可以积累。积累时间为15小时。 <img alt="图3" src="/files/ftp_upload/3895/3895fig3.jpg" /> 图3。增长尼特+和介质选择性MMTS NIT-殖民地之间的差异。 NIT的殖民地（箭头）较小。 NIT的殖民地（箭头），显示出强劲的增长。拍照后7天。

<ol>
	<li>Bowden, R. L., Leslie, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+recombination+in+Gibberella+zeae.">Sexual recombination in Gibberella zeae.</a> Phytopathology. 89, 182-188 (1999).</li><li>Buller, A. H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Researches on Fungi. , (1909).</li><li>Cavinder, B., Hamam, A., Lew, R. 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H., Proctor, R. H., Lee, T., Plattner, R. D., Lu, S. -. W., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+and+complementation+of+the+mating+type+(MAT)+locus+of+the+wheat+head+blight+pathogen+Gibberella+zeae.">Deletion and complementation of the mating type (MAT) locus of the wheat head blight pathogen Gibberella zeae.</a> Applied and Environmental Microbiology. 70, 2437-2444 (2004).</li><li>Gueldener, U., Seong, K. -. Y., Boddu, J., Cho, S., Trail, F., Xu, J. -. R., Adam, G., Mewes, H. -. W., Muehlbauer, G. J., Kistler, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Fusarium+graminearum+Affymetrix+GeneChip+For+profiling+fungal+gene+expression+in+vitro+and+in+planta.">Development of a Fusarium graminearum Affymetrix GeneChip For profiling fungal gene expression in vitro and in planta.</a> Fungal Genetics and Biology. 43, 316-325 (2006).</li><li>Hallen, H., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+L-Type+calcium+ion+channel+Cch1+affects+ascospore+discharge+and+mycelial+growth+in+the+filamentous+fungus+Gibberella+zeae+(anamorph+Fusarium+graminearum).">The L-Type calcium ion channel Cch1 affects ascospore discharge and mycelial growth in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum).</a> Eukaryotic Cell. 7, 415-424 (2008).</li><li>Hallen, H., Huebner, M., Shiu, S. -. H., Guldener, U., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+shifts+during+perithecium+development+in+Gibberella+zeae+(anamorph+Fusarium+graminearum),+with+particular+emphasis+on+ion+transport+proteins.">Gene expression shifts during perithecium development in Gibberella zeae (anamorph Fusarium graminearum), with particular emphasis on ion transport proteins.</a> Fungal Genetics and Biology. 44, 1146-1156 (2007).</li><li>Lee, J., Lee, T., Lee, Y. -. W., Yun, S. -. H., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shifting+fungal+reproductive+mode+by+manipulation+of+mating+type+genes:+obligatory+heterothallism+of+Gibberella+zeae.">Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae.</a> Molecular Microbiology. 50, 145-152 (2003).</li><li>Klittich, C. J. R., Leslie, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrate+reduction+mutants+of+Fusarium+moniliforme+(Gibberella+fujikuoi.">Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuoi.</a> Genetics. 118, 417-423 (1988).</li><li>Min, K., Lee, J., Kim, J. -. C., Kim, S. G., Kim, Y. H., Vogel, S., Trail, F., Lee, Y. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+gene,+ROA,+is+required+for+proper+morphogenesis+and+discharge+of+ascospores+in+Gibberella+zeae.">A novel gene, ROA, is required for proper morphogenesis and discharge of ascospores in Gibberella zeae.</a> Eukaryotic Cell. 9, 1495-1503 (2010).</li><li>Qi, W., Kwon, C., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+of+transcript+accumulation+during+perithecium+development+in+Gibberella+zeae+(anamorph+Fusarium+graminearum).">Microarray analysis of transcript accumulation during perithecium development in Gibberella zeae (anamorph Fusarium graminearum).</a> Molecular Genetics and Genomics. , 276-287 (2006).</li><li>Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+cannons:+explosive+spore+discharge+in+the+Ascomycota.">Fungal cannons: explosive spore discharge in the Ascomycota.</a> FEMS Microbiol. Lett. 276, 12-18 (2007).</li><li>Trail, F., Common, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perithecial+development+by+Gibberella+zeae:+a+light+microscopy+study.">Perithecial development by Gibberella zeae: a light microscopy study.</a> Mycologia. 92, 130-138 (2000).</li><li>Trail, F., Xu, H., Loranger, R., Gadoury, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+environmental+aspects+of+ascospore+discharge+in+Gibberella+zeae+(anamorph+Fusarium+graminearum.">Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum.</a> Mycologica. 94, 181-189 (2002).</li></ol>

我们什么都没有透露。

小麦赤霉病是特别适合子实体发展的研究，由于良好注释的基因组（可用性<a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/">mips.helmholtz-muenchen.de/genre/proj/FGDB /</a> 4），Affymetrix公司的可用性基于芯片6。这有助于识别能力，为子囊孢子放电7,11,3重 ​​要的基因。这里介绍的方法将允许研究人员关注的发展阶段和功能的一组离散楼子实体的遗传分析禾谷 。方法也很容易适应相关的真菌可以诱导水果文化，可以作为一个发展中的各种其他真菌子实体类型的标准。 能力评估孢子发射型孢子小，很难看到的物种，如可以是微妙<eM&gt; F。禾谷。一个干净的幻灯片孢子的收集有利于双方的视觉评估和定量评估，为孢子可以洗掉的幻灯片和定量分析。一些真菌物种产生一种粘液，围绕着他们的孢子，孢子不能被冲​​洗掉，但必须计算显微镜，他们留在幻灯片上。我们已经发现这个菌核病是真实的。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>禾谷镰刀菌菌株的PH-1 </td><td>真菌遗传学联合中心，堪萨斯</td><td> FGSC 9075 </td></tr><tr><td>吐温60 </td><td> Sigma-Aldrich公司</td><td> P1629 </td></tr><tr><td>查氏的阿霉素琼脂</td><td> Difco公司，碧迪</td><td> 233810 </td></tr><tr><td>氯酸钾</td><td> Sigma-Aldrich公司</td><td> 255572 </td></tr></tbody></table>

sexual development and ascospore discharge in fusarium graminearum

禾谷镰刀菌已成为一个模型系统开发和丝状真菌的致病性研究。 楼菌最容易产生子实体，称为子囊壳，胡萝卜琼脂。子囊壳含有大量的组织类型，在子囊发展的特定阶段产生。这些措施包括（按出场顺序）形成子囊缩写（上升到ascogenous菌丝），外墙，侧丝（无菌菌丝占据子囊中心，直到ASCI发展），子囊和子囊在ASCI 14。这些组织的发展，相隔约24小时，并一直在性发展12,8转录研究的基础上。哈伦等人 （2007年）的发展更深入的说明，包括每个阶段的照片。在这里，我们提出的方法，产生和收获同步LY发展子囊草坪时空的基因调控，发展和生理过程的研究。虽然这些方法是专门用于与楼菌 ，该技术可以用于其他各种真菌，规定可以培养诱导出菇，并有一定的发展同步。最近，我们已经适应了这个协议研究楼性发展verticillioides。虽然个别子囊必须手工采摘，在这个物种，因为发展中国家子囊壳的草坪，不能诱导，工作过程以及研究发展（Sikhakolli和步道，未发表）。 真菌子实体的最重要的功能是分散孢子。子囊菌门（子囊生产真菌），许多物种，射门从子囊孢子，由于代膨压力范围内的子囊，驾驶弹射孢子（和epiplasmic流体）通过在子囊尖端2,7孔。发展“孢子放电法”，这是我们使用的筛选过程中的突变导致我们强行子囊孢子放电的研究。在这里，我们提出这个实验的细节。 小麦赤霉病是homothallic，从而可以在兼容的合作伙伴的情况下形成的子实体。同宗配合的优势是隧道是没有必要的，以产生特定性状的后代，促进性发展的研究方面在这个物种14,7合子。然而，异宗株已产生可用于穿越5,9。它也可能跨越homothallic株，获得1株1中的几个基因的突变。这是通过一个培养皿coinoculating 2株。沿交汇点，子囊多数将重组（父株突变不抑制杂交）。子囊壳的年龄，他们散发出子囊集体，而不是强行履行。所产生的孢子渗出物（称为一个cirrhus）位于子囊尖和恢复单个孢子可以很容易地删除。在这里，我们提出了一个协议，以方便识别重组子囊壳和重组后代恢复。

Watch this Scientific Journal Video about Sexual Development and Ascospore Discharge in Fusarium graminearum at JoVE.com

Sexual Development and Ascospore Discharge in Fusarium graminearum

性杂交和重组后代的隔离是丝状真菌的重要研究工具，<em&gt;禾谷镰刀菌</em&gt;，必要的技术，成功地开展这些过程。

性发展和有性放电禾谷镰刀菌</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

sexual-development-and-ascospore-discharge-in-fusarium-graminearum

Research

JoVE Journal

Biology

Sexual Development and Ascospore Discharge in Fusarium graminearum

21.7K 次观看.  Michigan State University. 性杂交和重组后代的隔离是丝状真菌的重要研究工具，禾谷镰刀菌，必要的技术，成功地开展这些过程。

视频: Sexual Development and Ascospore Discharge in Fusarium graminearum

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae子囊孢子生产和收集微阵列实验。

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

科研

生物学

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

使用IVIS频谱的乳腺肿瘤在体内生物发光成像

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

结合DNA-RNA荧光<em&gt;原位</em&gt;杂交（FISH）研究X染色体失活的分化雌​​性小鼠胚胎干细胞

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

在活细胞悬浮液荧光测定G蛋白选择性GPCR构象测量使用FRET传感器

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...