The authors would like to acknowledge funding support for aspect of the work through Linkage Grants LP0776563 (GB, AS) and LP0211919 (GB) and industry partners Sappi and Mondi as well as Australian Postgraduate Award (EM) from the Australian Research Council and the Young Innovators and Scientist Award through the Australian Department of Agriculture (LT). We also like to thank the Zander Myburg, Qing Wang, Colleen MacMillan and Simon Southerton for the many discussions and ideas they put forward during the development of this protocol and to Martin Ranik, Minique De Castro, Julio Najera, Valerie Frassiant, Angelique Manuel and Noemie Defaix for assistance in laboratory related work.

1.植物材料的制备<ol><li>之前的实验，提高从种子或切割的优选树种新的幼苗和生长树/秒，直到在用于实验的区域中的杆的直径是直径约1厘米。 注:需要可能会有所不同，由于植物的生长速度，因此三至九个月这一步之间允许的时间。 </li></ol> 2.二进制向量创建<ol><li>从此部分进行工作，以在实验室或温室，其中根癌农杆菌可以处理和规定为实验室适当的个人防护设备部分7被使用。 </li><li>制备含有任何兴趣击倒或过表达盒和一个β葡萄糖醛酸酶（GUS）报道基因盒的一个基因或融合于取决于研究的类型GUS基因的感兴趣的启动子的二元载体。 注意:有一些二元载体BAC的可商购或可来源通过提供给插入基因和感兴趣的启动子进二元载体研究网络以及许多技术kbones。在这个协议的情况下达到实验者就如何创建一个二进制向量的决定。 </li><li>变换二元载体导入A的解除武装应变利用农杆菌电或热休克14，直到需要进行实验适当存放。 </li><li>重复步骤2.2为任何阳性和/或阴性对照。 注意:阴性对照应包括含有无目的基因进行兴趣研究的基因和感兴趣研究的启动子的启动子的GUS二元载体。利息研究的启动子阳性对照应包括融合到GUS记者花椰菜花叶病毒35S启动子（CaMV35S启动）。 </li></ol> 3. A.制备农杆菌接种<ol><li>至少一周之前EXPERimentation，拿A.少量（约1微升）步2.2和2.3 农杆菌中，并准备与琼脂独立LB培养基上显得捉襟见肘含有细菌选择适当的抗生素板块14。在孵化器成长在28°C，形成小菌落，然后在4个商店 直到需要°以下。 </li><li>之前的实验48小时后，转移A的一个菌落从各板成的50ml 农杆菌螺杆含有5ml预热（28℃）的LB培养基包含用于细菌选择适当的抗生素和在200rpm搅动振荡器上孵化约48小时，在28℃直至14的顶管混合物非常浑浊。 </li><li> 4-6之间茎小时之前，工厂的接种（第4）加1毫升LB / A的农杆菌混合成一个新的容量50mL的螺纹含有新鲜温热（28°C）24毫升（1:20稀释）顶管LB培养基与适当的抗生素细菌选择。如果在600nm（OD 600，如通过光谱学测定）的光密度（OD）大于0.1，直到这实现进一步用温水的LB稀释。 </li><li>将稀释LB / A.农杆菌混合重上摇动孵化和动摇在相同条件下（200转，28°C），直到OD 600为0.4-0.6，并删除之间。 </li><li>离心机LB / A.农杆菌混合物在1000 xg离心15分钟，4℃。 </li><li>倒出液体，并立即重悬A.农杆菌沉淀在1ml的冷却MS媒体15，转移〜2毫升离心管，直到需要接种（第4）冰上存储。这种溶液被称为接种媒体。 </li></ol> 4.接种植物与干A.农杆菌 <ol><li>春末或初夏使用具有积极而快速增长形成层在第1中创建植物在开始实验。一个好的诊断为活性和快速增长的形成层是韧皮部可从木质部容易地剥离。 </li><li>发现茎植物的基地附近一个清晰的直线部分，并移除任何树叶和树枝。 </li><li>用锋利的手术刀（最好是第11号）或刀片，通过引入两个垂直平行的切口，通过20毫米的长度和1个5毫米相隔韧皮部再创建茎明确的部分1厘米2&#39;形成层窗口"横向切断连接在其底端的两条垂直切割。 </li><li>通过剥离切口向上露出木质部发展组织及步骤3.6补充足够的接种媒体湿用吸管（通常是5-10微升）露出木质部开发的表面产生的韧皮部条。立即重新插入韧皮条。 </li><li>绑定韧皮部地带茎紧密封口膜。 </li><li>重复步骤4.2至4.5为任何额外形成层窗户兴趣创建的基因（S）或子（S）NY新形成层窗口至少1cm上方或下方等形成层的窗口，并在90°的偏移。 </li><li>完成步骤4.2至4.5的阳性和阴性对照载体（步骤2.4）确保每个向量被添加到在实验中使用的各植物的相同干。 </li><li>定期监测树干直径的生长和收获GUS检测时至少5 mm径向增长茎中被观察到（第5）。 注意:所需要的径向生长的量依赖于组织的用于下游分析所需的量。 </li></ol> 5.收获了GUS的组织学分析<ol><li>消费税从阀杆"形成层窗口&#39;移动任何组织不能形成层窗口中新的增长点的一部分。 <ol><li>对于与成熟的木质部和韧皮部的细胞/组织形态的研究，从剥离木质部韧皮部。 </li><li>对于需要在形成层区保持完整或启动子的表达模式的研究是要评估版，切片形成层窗口横向使用刀片或其他锋利的刀刃成厚0.5和1之间的光盘。 </li></ol></li><li>放置加工形成层窗口组织在14毫升圆底管和在pH 7 14 0.1M磷酸盐缓冲液冲洗两次。确保组织被完全淹没，保留在溶液中至少五分钟，所有过量的溶液在末端被除去第二漂洗。 </li><li>添加5毫升GUS试剂（0.5毫米×-GLUC（5-溴-4-氯-3-吲哚 - β-D-葡糖醛酸），10毫摩尔EDTA，0.5％的Triton X-100 V / V，0.5毫钾铁氰化物（III），0.05毫亚铁氰化钾（II）中，由于最终体积为0.1mM磷酸盐缓冲液pH 7;参见Hawkins 等 16）到每个管中。如果组织没有完全淹没，添加额外的GUS试剂。 </li><li>在黑暗中在55℃孵育管10分钟。 </li><li>在黑暗中在37℃孵育管用于在摇床培养箱进一步12-16小时使用（30至60转）温和搅拌以允许混合。 </li><li>当从摇床培养箱除去随机检查GUS试剂的pH值使用石蕊试纸pH范围0-7管的一小部分。 <ol><li>如果任何管子的pH值低于6再贴上标签。继续检查管子的其余部分，以确认pH和标签如果pH为6或更低。 注意:用pH值低于6的样品可能不适合用于分析。这些样品必须从进一步分析中排除。 </li></ol></li><li>滗GUS试剂并有足够的70％的乙醇代替以覆盖组织。 </li><li>直到需要储存于4℃。 </li></ol> 6.确定转基因组织<ol><li>使用镊子，取出全部来自于小托盘或培养皿管和地点形成层窗口组织允许微观可视化。 </li><li>使用1X和4X放大率之间的解剖显微镜，鉴定并计算细胞或组织的数量有一个明亮的蓝色染色。蓝染色的细胞或组织被称为从这时开始扇区。理货他们在以下几个方面。 <ol><li>对于其中韧皮部已被删除（步骤5.1.1）的样品，计算在显影木质部（形成层扇区， 图1a）的表面上发现形成层组织的扇区数。 </li><li>对于已经被切割成盘（步骤5.1.2）的样品，计算在不同的细胞或组织类型中发现的扇区数（ 图1B，1C，1D）。扇区可以在以下的组织类型中找到:周皮（周皮扇区， 图1E），韧皮部（韧皮部扇区， 图1F），形成层组织（形成层扇区， 图1G，1H，1I），伤口实质（伤口薄壁扇区， 图1J）和侵填体（TYLOSE部门， 图1K）。 <ol><li>在显微镜下一个ssessment确保盘的两侧已经被浏览和跨两个盘出现的行业相匹配，以使数字不被高估。 </li></ol></li></ol></li><li>放置仅含有组织部门放回含有70％的乙醇和存储，直到需要该管。 </li><li>重复步骤6.1至6.3为任何额外形成层窗口包括正和/或阴性对照。 </li><li>除以扇区的总数乘以1厘米2形成层窗口的总数来推导每形成层的cm 2的转化事件的平均数计算每个扇区类型为每个基因或启动子和控制平均转化效率分别接种（ATScm -2）。 </li><li>继续执行步骤7.2和8的启动子表达模式的分析和步骤7.1，7.2或7.3的技术评估组织形成层细胞和组织的形态。 </li></ol> 7.分析在形成层组织细胞和组织形态注:下面是已成功地用于ISSA的分析技术的选择衍生的样品。 <ol><li>分析木质部小区区域，管腔面积，细胞壁厚度和细胞壁面积使用扫描电子显微镜（SEM）的。 注:此协议需要最少的样品制备和允许相对高吞吐量与概括的形态性状行业分析。 <ol><li>从这一步开始，请确保白大褂，保护眼睛和木本时组织被处理，处理后用于手套。 </li><li>识别用于分析的形成层部门和使一个切口大约0.5mm至其任一侧使用单个边缘刀片创建光盘（ 图2a）。 </li><li>修剪掉多余木质部组织留下木质部组织约1mm到扇区的切向一侧（ 图2b）。 </li><li>小心LY使用新鲜双刃刀片（ 图2c）横向切割通过该扇区的中心。 </li><li>使在横向平面任扇区侧的两个附加浅径向切口划定转基因组织（ 图2d）。如GUS染色不会深入渗透到组织沿着细胞的径向文件遵循在形成层表面发现染色的组织的两侧。 </li><li>附上准备形成层界面到的SEM销存根的阶段使用SEM导电性带（ 图2e）装入和保持在干燥器直到需要用于SEM可视化。 </li><li>重复步骤7.1.2至7.1.6为任何附加扇区包括来自阴性对照。 </li><li>在低真空模式使用SEM（能源5千伏，斑3.0纳米），可视化和取该扇区内的细胞/组织的照片，以及将细胞/组织直接相邻的扇区（ 图2F）。倍率的量依赖于感兴趣的特征。对于木质部纤维，是2000倍适宜（ 图2G）。 </li><li>一旦形成层部门已经显现，并在适当的放大倍率拍摄的照片，用图像测量软件测量感兴趣木质部细胞/组织形态特征。 </li><li>进行统计分析，并进行多成对分析使用配对t检验来比较在转基因部门和相邻的非转基因的对照细胞/组织用于每个扇区确定的p（内从扇区边缘0.5毫米测量）测量形态之间的差-值。 </li><li>重复步骤7.1.10为阴性对照。 <ol><li>在阳性对照示出了低的p值（α&lt;0.05）的情况下，计算出转基因和相邻的非转基因细胞/组织的形态学性状之间的差异。在非配对t检验使用这个"差分值"与negat比较感兴趣的基因的作用香港专业教育学院控制以确定p值。 </li></ol></li></ol></li><li>使用光学显微镜的干细胞/组织的形成层细胞/组织形态或启动子表达模式组织化学分析。 注:虽然耗费更多的时间，这种方法允许更多的分析选项，包括形成层动态， 如形成层宽度以及启动子表达模式的各个方面。此外，如果无法访问的SEM，该协议可被用作用于步骤7.1的替代品。 <ol><li>使用剃刀刀片兴趣消费部门和直接与螺丝帽在至少2天的样品小瓶1-3毫升100％的乙醇于4℃一些周围组织的小块（不大于1mm 3）和地点在摇床上。的方式，将允许感兴趣的表面，以用切片机进行访问准备小块。 </li><li>重复为任何附加扇区包括来自阴性对照。 </li><li>除去液体并用2取代5％乙醇:75％的LR白混合物2天维持的条件。 </li><li>重复步骤7.2.3用50％乙醇:50％的LR白，25％乙醇:75％的LR用100％LR白白然后最后两次。 </li><li>将小块与对齐到短端利率表面嵌入模具，并用新鲜LR的白色仔细覆盖和聚合按照制造商的说明。 </li><li>切割在旋转切片机从感兴趣的表面5μm的部分，并安装在载玻片。用番红O（0.01％）染色可视化细胞壁和/或其它特征。 </li><li>添加安装媒体，放置盖玻片，并允许设置过夜。 </li><li>查看100X和600X放大倍率和捕捉图像的光学显微镜下。 </li><li>从捕获使用图像测量软件图像形态特征。 <ol><li>对于定量形态性状的统计分析，从步骤7.1.10遵循。 </li><li>对于形态性状的定性分析，比较和描述了基因或感兴趣的启动子和负极和/或阳性对照观察到的模式。 </li></ol></li></ol></li><li>在浸软纤维使用光学显微镜微纤丝角（MFA）分析。 <ol><li>消费转基因木质部直接从一个扇区以及从邻近它的非转基因组织的组织（在0.5毫米， 图2h），并发生在分开1.5毫升管中。重复为任何附加扇区包括来自阴性对照。 </li><li>完成步骤7.3.3 7.3.5在通风橱。 </li><li>添加过氧化氢的250微升和250微升冰醋酸至每个管。 </li><li>放置管在加热块在90℃下，在通风橱2小时。 </li><li>从管中取出组织并用蒸馏水仔细漂洗至少两次。 </li><li>使用的水溶性安装介质，安装在载玻片组织并与前固定用盖玻片尖锐的镊子挑逗分开。 </li><li>视图下，在放大倍数超过400倍较大的个体纤维的光镜和捕捉图像。 </li><li>为了确定纤维的微纤丝角，测量坑孔和/或细胞壁条纹和利用图像测量软件的纤维（ 图2I）的长轴之间的角度。 </li><li>进行统计分析，从步骤7.1.10遵循。 </li></ol></li></ol> 8.启动子的分析表达模式<ol><li>分析专干组织中启动子表达模式。 <ol><li>相符的每个扇区类型感兴趣的启动子，以及阳性和阴性对照的频率如在步骤6.2.2说明。 </li><li>比较不同扇区类型的频率的使用卡方检验，建立p值的阳性对照感兴趣的启动子。细胞/组织特异性的进一步分析可以使用步骤7.2根据需要进行。 <ol><li>如果多于一个的启动子的兴趣正在调查然后重复所有感兴趣的启动子和阳性对照之间的这种分析作出比较。 </li><li>如果阴性对照观察到的部门或蓝染色，然后将它添加到分析，或根据程度或内源性染色怀疑（见讨论 ）抛弃。 </li></ol></li></ol></li><li>启动子表达模式的分析过程中形成层衍生发展与分化。 <ol><li>使用解剖显微镜，可视化所有在步骤6.2中确定的形成层的扇区（图1G，1H，1I），并判断在从形成层的三种组织类型的存在/不存在蓝染色的;韧皮部（P），显影木质部（X1）或开发的木质部组织（X2）（参见图3a）。 </li><li>按照步骤8.1.2，比较存在/不存在GUS染色的频率中的int启动子的P，X1和X2的区域用卡方检验，建立p值阳性对照erest。细胞/组织特异性的进一步分析可以使用步骤7.2根据需要进行。 <ol><li>欲了解更多考虑的步骤8.1.2.1 8.1.2.2和。 </li></ol></li></ol></li></ol>

<ol>
	<li>Spokevicius, A. V., Tibbits, J. F. G., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+plant+and+plant+part+transgenic+approaches+in+the+study+of+wood+formation+-+benefits+and+limitations.">Whole plant and plant part transgenic approaches in the study of wood formation - benefits and limitations.</a> TPJ. 1 (1), 49-59 (2007).</li><li>Chaffey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wood+formation+in+forest+trees:+from+Arabidopsis+to+Zinnia.">Wood formation in forest trees: from Arabidopsis to Zinnia.</a> Trends Plant Sci. 4 (6), 203-204 (1999).</li><li>Moller, R., McDonald, A. G., Walter, C., Harris, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+differentiation,+secondary+cell-wall+formation+and+transformation+of+callus+tissue+of+Pinus+radiata+D.+Don.">Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D. Don.</a> Planta. 217 (5), 736-747 (2003).</li><li>Spokevicius, A. V., Van Beveren, K., Leitch, M. M., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+in+vitro+transformation+of+wood-producing+stem+segments+in+eucalypts.">Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts.</a> Plant Cell Rep. 23 (9), 617-624 (2005).</li><li>Plasencia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eucalyptus+hairy+roots,+a+fast,+efficient+and+versatile+tool+to+explore+function+and+expression+of+genes+involved+in+wood+formation.">Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.</a> Plant Biotech J. , (2015).</li><li>Van Beveren, K. S., Spokevicius, A. V., Tibbits, J., Wang, Q., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+cambial+tissue+in+vivo+provides+efficient+means+for+Induced+Somatic+Sector+Analysis+(ISSA)+and+gene+testing+in+stems+of+woody+plants+species.">Transformation of cambial tissue in vivo provides efficient means for Induced Somatic Sector Analysis (ISSA) and gene testing in stems of woody plants species.</a> Funct Plant Biol. 33 (7), 629-638 (2006).</li><li>Spokevicius, A. V., Van Beveren, K., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+transformation+of+dormant+lateral+buds+in+poplar+trees+reveals+developmental+patterns+in+secondary+stem+tissues.">Agrobacterium-mediated transformation of dormant lateral buds in poplar trees reveals developmental patterns in secondary stem tissues.</a> Funct Plant Biol. 33 (2), 133-139 (2006).</li><li>Spokevicius, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-tubulin+affects+cellulose+microfibril+orientation+in+plant+secondary+fiber+cell+walls.">beta-tubulin affects cellulose microfibril orientation in plant secondary fiber cell walls.</a> Plant J. 51 (4), 717-726 (2007).</li><li>MacMillan, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fasciclin-like+arabinogalactan+protein+family+of+Eucalyptus+grandis+contains+members+that+impact+wood+biology+and+biomechanics.">The fasciclin-like arabinogalactan protein family of Eucalyptus grandis contains members that impact wood biology and biomechanics.</a> New Phytol. 206 (4), 1314-1327 (2015).</li><li>Creux, N. M., Bossinger, G., Myburg, A. A., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+somatic+sector+analysis+of+cellulose+synthase+(CesA)+promoter+regions+in+woody+stem+tissues.">Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues.</a> Planta. 237 (3), 799-812 (2013).</li><li>Hussey, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SND2,+a+NAC+transcription+factor+gene,+regulates+genes+involved+in+secondary+cell+wall+development+in+Arabidopsis+fibers+and+increases+fiber+cell+area+in+Eucalyptus.">SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibers and increases fiber cell area in Eucalyptus.</a> BMC Plant Biology. 11, (2011).</li><li>Melder, E., Bossinger, G., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+ARBORKNOX1+delays+the+differentiation+of+induced+somatic+sector+analysis+(ISSA)+derived+xylem+fiber+cells+in+poplar+stems.">Overexpression of ARBORKNOX1 delays the differentiation of induced somatic sector analysis (ISSA) derived xylem fiber cells in poplar stems.</a> Tree Genet. Genomes. 11 (5), (2015).</li><li>Baldacci-Cresp, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PtaRHE1,+a+Populus+tremula+x+Populus+alba+RING-H2+protein+of+the+ATL+family,+has+a+regulatory+role+in+secondary+phloem+fiber+development.">PtaRHE1, a Populus tremula x Populus alba RING-H2 protein of the ATL family, has a regulatory role in secondary phloem fiber development.</a> Plant J. 82 (6), 978-990 (2015).</li><li>Sambrook, J., Russell, D. 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> Molecular cloning: A laboratory manual. , (2001).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio-assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio-assays with tobacco tissue cultures.</a> Physiol. Plant. 15 (3), 473-497 (1962).</li><li>Hawkins, S., Pilate, G., Duverger, E., Boudet, A., Grima-Pettenati, J., Chaffey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+GUS+histochemistry+to+visualise+lignification+gene+expression+in+situ+during+wood+formation.">The use of GUS histochemistry to visualise lignification gene expression in situ during wood formation.</a> Wood formation in trees: Cell and Molecular Biology Techniques. , 271-295 (2002).</li><li>Hodal, L., Bochardt, A., Nielsen, J. E., Mattsson, O., Okkels, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+expression+and+specific+elimination+of+endogenous+beta-glucuronidase+activity+in+transgenic+and+nontransgenic+plants.">Detection, expression and specific elimination of endogenous beta-glucuronidase activity in transgenic and nontransgenic plants.</a> Plant Sci. 87 (1), 115-122 (1992).</li><li>Hansch, R., Koprek, T., Mendel, R. R., Schulze, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+for+eliminating+endogenous+beta-glucuronidase+background+in+barley.">An improved protocol for eliminating endogenous beta-glucuronidase background in barley.</a> Plant Sci. 105 (1), 63-69 (1995).</li></ol>

The authors declare that they have no competing financial interests.

在ISSA协议是在几个月的基因和所涉及的木材感兴趣的启动子的分析在空间中树种创造转基因茎组织的相对简单和有效的方法和茎的形成。举手之劳，除了保持植物活，需要成长接种后其鲜明的对比，其中需要大量的培养，以维持组织或植物，其中木材生产可能需要长达几年开始或者一个真正的体外方法转基因茎组织专干不创建1。 ISSA也被证明是适用于广泛范围的树种，包括杨树...

树茎包括行星生物量的显著量是巨大的生物，文化和商业上的重要性。二级茎由许多其他生命形式提供资源和庇护所创造的栖息地。他们以他们居住，并充当生产木材，纸浆和造纸等木材和非木材产品的可再生资源的生态系统提供多项服务。二次干发展并且更具体木材形成通过调节特定细胞类型的发展复杂的分子体系的约束，其细胞壁的生化成分，以及他们如何被布置为形成组织和器官。解剖专干的开发和木材形成的分子基础是由许多因素，包括木材和干性内部和之间的茎，长代次，异交交配系统，高杂合度，高遗传负荷，季节性休眠，长的可变性混淆成熟特点建立期和成熟的树木纯粹的物理尺寸。其结果是，干的发展相对于植物发育的分子控制的其它方面的详细知识次要的认识，仍处于起步阶段。 许多体外技术已被用于研究和了解二次干发展，尤其是木材和二级细胞壁的形成。这些协议包括使用整个植物或植物部分的系统，其中任一转基因植物产生或特定辅助细胞或组织转化为木材和/或仲干发展1的具体方面的研究。转基因植物可以从各种各样的植物组织和细胞类型的回收后的遗传转化，但是，进展缓慢，特别是在分析木纤维性状由于长期再生时和干成熟时间（年的顺序），高的技术和劳动德曼DS，可以通过低的候选基因，以及在传播一些木本植物物种困难。类似的技术已在非木质模型系统被开发，如拟南芥，成功地克服了这些限制，但不是所有的二次干细胞类型中在这些本茎和相关的季节性或长寿性状不能在这些物种2进行研究。备选地，植物部分系统，例如Pinu酒店小号辐射愈伤组织培养物3减少相关的时限。然而这些方法被限制于单个细胞类型的研究，并如所指出用于体外实验遭受类似的限制。同样，涉及整个茎段根尖干文化4显示的承诺，但目前还没有适用于特定基因或发起人的研究。最近，涉及毛状根替代协议已被开发用于桉树并已成功施加5，但是，这种方法仍需要在体外培养，涉及二次根而非茎和迄今它仅限于单一树种。 诱导的体部门分析（ISSA），如这里描述的，被开发来克服的一些问题，提供用于基因和启动子的高通量功能筛选工具的培养基中，在木材形成和二次茎组织发育疑似角色。 ISSA是被开发，以减少所产生的转基因细胞和组织在一个完整的辅助阀杆同时克服劳动时间在体内转化和筛选系统，体外方法中使用的常规技术和吞吐量的限制。这里所描述的协议允许用于同时建立数百独立地转化组织扇区和小区的一个短的时间期间内的次级茎，在树种和感兴趣组织而不克相对较短的时间框架和低廉的劳动力成本中enetic和/或环境的变化。 ISSA 体内技术首先被用于辅助阀杆6和芽7组织以来，通过参与形成层分化和基因和/或促进研究二次茎组织了细化说明，并有包括: 微管蛋白 （TUB）8，fasciclin样阿拉伯半乳聚糖 （ FLA）9， 纤维素酶 （CESA）10， 次生壁相关NAC域 （SND 2）11，ARBORKNOX（ARK1）12和非常有趣的新基因 （RING）H2蛋白13。在次级进行了这些研究茎杨树和桉树植物和提供见解细胞形态，细胞壁化学和基因表达。 这里描述的协议的目的是汇集经验第二，通过知识的ISSA从一系列在过去十年中发表和未发表的研究开发和应用获得的。他们专注于专干组织6 在体内转化，集中力量对涉及新疆杨"锥状&#39;克隆， 蓝桉以及11个桉×赤桉克隆研究。本文通过从植物和细菌，茎组织，生长和组织的收获，转基因细胞和组织的鉴定，为收集和分析数据的表型评估和方法编制转型的种植协议进行研究。虽然技术已成功应用于测量细胞壁的单糖组成也9,11，由于篇幅所限，本文集中用于二次ST测量细胞和组织的形态和理解基因表达模式技术只有EMS。因此，如所概述的协议是适合于那些希望获得进一步深入了解的基因连接于次级的作用和/或表达的茎使用成本低，在技术上容易，中到高通量方法。

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Plants</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">Please consult local nursery suppliers for plants as needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Agrobacterium strain</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">There are many possible avenues to obtain Agrobactrium strains. We suggest you follow up within your local research community as there may be restrictions in obtaining the bacteria in your country and region.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:188px;">Binary vector (gene and promoter)</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">We have developed a range of vectors to suite the ISSA protocol using a the Gateway Recombinase system. This include overexpression, RNAi knockouts and promoter fusion vectors based on modified pCAMBIA vectors and happy to provide as needed. In addition, there are many vectors avialable to the research community.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LB media</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">L3022</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LB media with agar</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">L2897</td>
			<td style="width:751px;">A like product could be sourced from another company</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Antibiotics</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">The catalog number will be dependent on the antibiotic you require as a range of antibiotic are used for bacterial selection in binary vectors. This product could be sourced from a&#160; range of companies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">50 ml Screw top tubes</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">2 ml Microtube</td>
			<td style="width:95px;">Watson Bio Lab</td>
			<td style="width:119px;">132-620C</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">MS Media</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">M9274</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Scalpel blade no 11</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">S2771</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Parafilm &#34;M&#34;</td>
			<td style="width:95px;">Bemis</td>
			<td style="width:119px;">PM996</td>
			<td style="width:751px;">This is the best product to use to bind the cambial window post creation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">14 ml round bottom tubes</td>
			<td style="width:95px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">150268</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">EDTA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Triton</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">X-Gluc</td>
			<td style="width:95px;">X-GLUC direct</td>
			<td style="width:119px;"></td>
			<td style="width:751px;">You will need to go to the website to order - http://www.x-gluc.com/index.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Potassium Ferricyanide (III)</td>
			<td style="width:95px;">Sigma</td>
			<td align="right" style="width:119px;">244023</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Potassium Ferrocyanide (II)</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">P9387</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Litmus paper</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">WHA10360300</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Single edge razor blade</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L055</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Double edge razor blade</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L056</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">SEM Pin Stub</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">GTP16111</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Sample vial with screw cap</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L6204</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Ethanol</td>
			<td style="width:95px;">sigma</td>
			<td style="width:119px;">E7023</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LR white</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">C025</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Embedding Mould</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">RL090</td>
			<td>We recommend this variety, however there are plenty of options available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Water Soulable mounting media</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">IA019</td>
			<td>One example of a mounting media that could be used however other options do exist and could be explored.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Hydrogen peroxide</td>
			<td style="width:95px;">Sigma</td>
			<td align="right" style="width:119px;">216763</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Glacial acetic acid</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">A9967</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Safranin O</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">C138</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:188px;">Quanta Environmental Scanning Electron Microscope</td>
			<td style="width:95px;">FEI</td>
			<td style="width:119px;"></td>
			<td>This is the instrument used at part of this study but any other SEM that has a low vacuum mode could be utilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Image J imaging software</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;can be sourced from the following URL http://rsbweb.nih.gov/ij/</td>
		</tr>
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the use of induced somatic sector analysis (issa) for studying genes and promoters involved in wood formation and secondary stem development

在树上二级茎生长和相关的木材形成都来自生物和商业的角度显著。然而，相对知之甚少的支配其发展的分子控制。这部分是由于经常与二次生长过程的研究相关的物理，资源和时间的限制。许多体外技术已被用于涉及两个木质和非木质植物物种或植物部分或整个植物系统。然而，关于它们的适用性进行二次茎的生长过程的研究问题，某些物种和劳动强度的顽抗往往望而却步中高吞吐量的应用。此外，在寻找专干发展，木材形成，当被调查的具体特征可能只历经数年的增长成为衡量一棵树的生命周期的后期。在解决体内 p这些挑战替代rotocols被开发出来，命名为诱导体部门分析，涉及直接在工厂的专干转基因体组织部门的创建。该协议的目的是提供一种有效的，简单和相对快速的方法来创建用于基因和启动子的功能分析，可在一个范围内树种可以利用转基因植物次生组织中。这里提出的结果表明，转基因次级干扇区可以在所有活的组织和细胞类型中创建二次茎的各种树种的木材形态特征的以及在次要启动子表达模式茎可容易地评估促进中到高通量功能特性。

使用该协议的所有活二次干细胞和组织类型已被证明是容易A.农杆菌转化并已确定为根据最初转化的细胞类型行业类型和其后续发展的增长模式。行业类型包括周皮，韧皮部，形成层，伤口实质和纤基乙酸钠（ 图1b，1C，1D）和一致的位置将在本段的剩余部分描述可以发现。一个周皮扇区包括发现只在周皮和从不延伸到所述韧皮?...

Watch this Scientific Journal Video about The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development at JoVE.com

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

诱导体细胞行业分析（ISSA）的为研究木材形成和二次开发茎相关基因和推动者使用

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

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Research

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Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

8.7K 次观看.  The University of Melbourne. Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

视频: The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

一种基于阵列的比较基因组杂交平台, 有效检测快中子诱导的苜蓿蒺藜突变体的拷贝数变化

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

qkat: 杀手细胞免疫球蛋白样受体基因的定量半自动分型

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.