This work was supported by the Ogelbay fund.

1.成长的植物<ol><li>前6周浸渍，填补5寸盆带土和母猪亚麻种子¼在土壤英寸深（每盆4种子）。一定要坚定了土超的种子。定期给植物浇水，保持它们长日照（14小时光照和10小时黑暗）。 </li><li>检查植物定期为基层花序（花簇"器官）。的植物都准备用于转化时，芽是可见的，刚刚形成在花序（ 图1和2）。看芽，必要时切断周围的树叶。 注意:使用最好的花期是至关重要的，并在讨论中详细说明。 <ol><li>使用植物的主要分支，因为它会产生更多的花比侧设有分公司的实验性治疗。使用侧分支或者作为对照组（不被浸渍），或用于其它实验处理。 </li><li>或者，使用的主，侧枝同一种植物的实验性治疗，并使用另一种植物作为控制或其他实验性治疗。使用标签来标记各个分支和日期和治疗型个人花。 </li></ol></li></ol> 2.克隆和转化的大肠杆菌 （ 大肠杆菌 ）细胞<ol><li>克隆感兴趣的片段/小基因导入植物二元载体携带在T-DNA的多克隆位点。 <ol><li>在一个步骤或两个步骤进行克隆。 <ol><li>直接克隆小刀片在这个协议（〜500 bp）的进厂二元矢量。在这个协议中，使用植物双载体（PRI909）。否则，使用任何其他植物的二元载体类似的策略。 </li><li>可替代地，克隆的大插入物（≥6.5KB）在两个步骤:首先使用一般商业克隆试剂盒（未种植特定的），然后亚克隆到植物二元载体。 </li></ol></li></ol></li><li>设立的PCR REAC灰以扩增感兴趣的基因。设计的引物在5&#39;端的限制性位点（根据所使用的植物二元载体的多克隆位点）。 <ol><li>使用标准的PCR基因组DNA的亚麻执行与以下循环条件:24℃的初始保持，然后2分钟，在94℃，随后在98℃的30个循环持续5秒，60℃15秒，并72℃2分钟。在72℃进行5分钟，随后无限期保持在4℃下进行最后的延伸步骤。 </li><li>于1％的Tris /硼酸盐/ EDTA（TBE）琼脂糖凝胶上分离PCR产物，运行在100伏的凝胶1小时。从凝胶中纯化的产物，并通过的NanoDrop量化。 </li></ol></li><li>建立连接混合物克隆PCR产物在商业克隆载体中。按照制造商的协议。 </li><li>变换连接混合物进化学感受态大肠杆菌细胞如下。 <ol><li>添加2微升的连接混合物的进入通道中的一个小瓶emically主管E.大肠杆菌细胞。在冰上孵育30分钟。 </li><li>热休克细胞30秒，在42℃（热休克的时间和温度取决于所使用的细胞类型）。 </li><li>在冰上，并加入250微升的RT与代谢物阻遏（SOC）中的超级最佳肉汤。 </li><li>盖上管并以200rpm孵育在轨道摇床在37℃下1小时。 </li><li>蔓延10 - 从上一个预热LB平板（预热，在37℃）+适当选择性抗生素每个转化50微升（确定基于使用的商业的克隆载体的类型，选择性抗生素）。孵育板在37°CO / N。 </li></ol></li><li>挑〜10个菌落为微型制备质粒纯化，使用商业试剂盒。赠送制备质粒纯化通常进行如下。 注:缓冲区名特定使用的商业工具包，但他们的一般功能是相似的。 <ol><li>在接种2的单菌落- 5ml的LB含有适当选择性抗生素（确定基于所使用的商业载体的类型）平台。 </li><li>孵育约8小时，在37℃剧烈振荡（〜300 RPM）。 </li><li>收获细胞，离心，在6000×g离心10分钟。 </li><li>悬浮在250微升悬浮缓冲沉淀。混合和涡旋以完全分散的颗粒。 </li><li> 6次 - 裂解的细菌细胞中加入250微升裂解缓冲液中，通过反相管4调匀。 </li><li>中和裂解物中的中和缓冲液并倒转4 - 6次以混合。离心在〜17900 XG 10分钟在台式离心。 </li><li>从前面的步骤，一个离心柱转移上清液。离心机再次〜17900 XG为30 - 60秒。 </li><li> 60秒 - 通过增加0.75毫升洗涤液和离心机30冲洗离心柱。丢弃的流量通过。 </li><li>离心机额外1分钟至字符型已经残留洗涤液。将离心柱在一个干净的1.5ml微量管中。为了洗脱DNA，加50微升水中，每列的中央。静置1分钟，并离心1分钟。 </li></ol></li></ol> 3.分析了纯化的质粒为插入体的存在<ol><li>限制精华: <ol><li>设置一个限制性消化消化与用于克隆的插入物的限制性内切酶的质粒以确定插入物的存在。一个典型的双限制性消化反应如下:1微克纯化的质粒，1μl的各限制酶，2微升10X限制性消化缓冲液+ 2微升BSA（如果适用的话）中，X微升水（总共20微升） </li><li>轻轻混匀，并培育在推荐的温度（从一个不同的酶另一个）。 </li><li>分析限制性酶切1％TBE琼脂糖凝胶反应，在100 V 1小时运行，并寻找辍学了适当的大小。 </li></ol></li><li> PCR分析: <ol><li>设立的PCR与纯化的质粒，使用引物从商用矢量区域以扩增插入物或载体和插入件之间的交界处的内部。通用引物是M13正向和反向和T3 / T7引物。 </li><li>在这个协议中，使用下面的PCR循环条件为:24℃，然后用2分钟，在94℃，随后18个循环的98℃下5秒，60℃15秒和72℃的初始保持2分钟。在72℃进行5分钟，随后无限期保持在4℃下进行最后的延伸步骤。 </li><li>在1％TBE琼脂糖凝胶负载的PCR产物和运行为100 - 120伏1小时。 </li></ol></li><li>排序: <ol><li>发送纯化质粒到商业测序工具来分析和确认插入物的存在。 </li><li>一旦获得正确的构建物，将其用于克隆的第二个步骤（到植物二元载体）。 注意: 步骤2.3 - 3.3如果克隆是在一个步骤中完成的，可以消除。 </li></ol></li></ol> 4.克隆到植物双元载体（PRI909）和E.大肠杆菌转化<ol><li>设置一个双限制性消化反应线性化植物二元载体，并使用加入到引物（ 步骤2.2）的相同限制性酶位点隔离先前克隆的插入物。 </li><li>分析使用凝胶电泳，100 V 1运行限制性消化 - 2小时。切割插入物，并从该凝胶线性植物二元载体。 </li><li>使用商业试剂盒来纯化的凝胶产品。参阅制造商的手册。 </li><li>建立一个连接反应结扎插入到植物二元载体。插入到载体的比例取决于该插入件的尺寸和植物二元载体。在这个协议中的LIS1插入了6.5 kb和植物二元矢量是9 KB。因此，用1:2插入到载体比例。 </li><li&gt;重复步骤2.4 - 3的细菌转化，质粒纯化和分析。一旦获得正确的构建物（插入+植物二元载体），则继续执行电穿孔，在步骤5。 </li></ol> 5.电穿孔进入农杆菌电感受态细胞<ol><li>解冻的农杆菌的小瓶农杆菌电感受态细胞在冰上。 </li><li>添加1纳克的二元载体质粒DNA到20微升感受态细胞，冰，轻轻混匀。 </li><li>冰浴一0.1厘米电比色皿。 </li><li>主管细胞/ DNA混合物转移到电试管，挖掘，收集底部混合物。放的反应杯在电穿孔机和脉冲（电压和时间条件取决于反应杯的大小和所使用的电穿孔）。 </li><li>加入1毫升SOC培养基和转移细胞到15ml管中。 </li><li>孵育1小时，在28 - 30°C，晃动在100转。板50 - 100微升细胞在LB琼脂+适当选择性抗生素的（取决于植物二元质粒和农杆菌中使用的菌株在这个协议中使用50μg/ ml的卡那霉素和100μg/ ml链霉素）。 </li><li>孵育平板达48小时，在28 - 30℃。 </li><li>菌落选择和质粒纯化重复步骤2.5。 </li><li>重复步骤3来分析的质粒，并使用该构建为花饰-浸渍之前，必须检查插入物和T-DNA的完整性。重复步骤3.2，在整个插入区域和整个T-DNA区和通过测序使用多个PCR引物如在步骤3.3。 注意:在这个协议中4种不同的引物，在整个T-DNA以及横跨刀片10的引物而设计的。 </li><li>一旦在选定的农杆菌菌落植物二元质粒的存在得到确认，地方小区iñ50％甘油，并储存在-80℃。商店剩余菌落在板在4℃下，如果它们被一个7星期内使用。 </li></ol> 6.花倾注:前2天，花卉浸渍: <ol><li>成长农杆菌细胞至平稳期（OD 600 0.5 - 1是可接受的）在LB +适当的抗生素在液体介质。 注意:这些是相同的抗生素如在步骤5.6，基于植物的二元载体和所用的农杆菌菌株的类型。 </li><li>开始培养以1:100稀释的饱和（5毫升）中的O / N培养并生长24 - 48小时，在28-30℃下以150rpm摇动。培养应已达到midlogarithmic相位和更可能会接近或在静止的阶段1。大约0.8 OD（0.5比1再度OD都可以接受）8。离心在5,000×收集细胞在RT克。 </li><li>悬浮细胞浸润在培养基（5.0％蔗糖+ 0.05 - 0.003％的Silwet L-77）。对于第一轮浸泡，用0.05％的Silwet-77。对于第二轮和第三轮，减低浓度至0.03％（以讨论详述）。 </li><li>继续花香浸一步。莱在其一侧的工厂，并在渗透中1-2分钟只沾可见芽。离开植物在其一侧并用保鲜膜覆盖它，以保持高湿度的圆顶（ 图3）。 </li><li>第二天，将植物在直立位置和正常维护。 </li><li>当芽变大（通常在约10 - 14天），重复步骤6.1 - 6.4。减少浸渍时间30 - 60秒的Silwet-77浓度为0.003％。 </li><li>维持厂一般直到它们的种子成熟，并准备被收集（ 图4）。 </li></ol> 7.选择积极的茶nsformants与直接PCR <ol><li>母猪在土壤中的T1种子按照步骤1.1。 <ol><li>可替代地，通过将​​2.2克的MS培养基+4克琼脂在500ml水中使Murashige和Skoog基础盐培养基（MS培养基）。高压釜中，倒入小花盆。保持在4℃直至使用。 </li><li>通过将其放在凝固MS培养基上播下种子。不断长日照（14小时光照和10小时黑暗）。种子将发芽在大约4 - 6天（ 图5）。 </li><li>从每朵花为出发点，用一个种子。如果没有获得阳性转化体，通过拾取从花另一个种子重复此步骤。 注:从同花测试不同的种子，因为在某些情况下，不能从一朵花所有的种子将被改造。从实验的花选择附加种子有时是必要的。 </li></ol></li><li>检查定期苗。在大约10 - 发芽后14天，当真实的离开发展，直接PCR测试工厂。 </li><li> 3叶 - 通过削减2准备的叶提取物的DNA - 从每苗（5毫克10重量），并放置在离心管。 </li><li>加入180微升的50mM的NaOH到每个管中，并孵育10分钟，在95℃下。 </li><li>通过加入20微升的1M的Tris-HCl（pH 8.0）中的中和的提取物。 </li><li>使用1微升提取物中的直接PCR反应中，使用在整个T-DNA或插入（ 图7）设计的引物，以选择阳性转化体。 <ol><li>在这个协议中，使用直接PCR为24℃的最初的保持，然后2分钟，在98℃，随后的（98℃40个循环持续10秒，15秒的退火步骤，并在68℃的延伸步骤C）。在68℃下5分钟，随后无限期保持在4℃下进行最后的延伸步骤。 （参考表1的详细信息，引物，退火温度和延长时间周期）。 </li></ol></li><li>确定阳性转化和他们成长到成熟。如果种子发芽，在MS培养基中，移植到土壤中一个更大的锅（ 图5）。 </li></ol>

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The authors have nothing to disclose.

在一些植物物种，如亚麻（ 亚麻 ），成功的植物转化受到了限制。以前，在亚麻转型需要农杆菌感染伤人及共培养，将生物射弹粒子或使用超声波超声处理，随后再生;而伴随着许多突变事件过程既漫长又容易。此外，这些技术的选择过程需要使用抗生素的选择标记如卡那霉素。然而，已经注意到在该选择的该方法产生许多假阳性，如亚麻趋于逃避高浓度的抗生素6,9,14文献。亚麻改造以前的技 ​​术的另一个缺点一直是低利率转变2,6。 在这里所描述的协议，经由花香浸渍农杆菌介导的植物转化中所示导致高转化率亚麻（50％ - 60％）。转化是从蘸花，收集来自主，侧枝获得。选择阳性转化只不过是通过种植对土壤T1植物和筛选它们的叶子发芽，他们不久后，绕过使用抗生素选择的做，一步步以前用作花香浸有模有其他植物物种。通过进行叶片直接PCR检测，并使用适当的T-DNA引物，阳性转化体可以迅速地选择。这种技术是简单，便宜且易于操作，但结果在高得多的转化率比以前用于拟南芥和其他植物物种报道使用这种方法1,10,12。它也是最高的报告的转化率为亚麻。 但是，也有在该程序的关键步骤包括最好花阶段的选择和最佳的表面活性剂浓度，以便农杆菌可以渗透到植物细胞而不杀死花器官。如果早期萌芽阶段时（ 图2A）具有高的Silwet-77浓度超过0.05％，则花不会发展，也不能设置的种子。如果后期芽阶段被使用（ 图2C），虽然变换可能工作，它将出现在低得多的速率。类似的结果用拟南芥花浸染变换1,4获得。此协议中，所有的花香阶段进行了测试与不同的Silwet-77的浓度和最佳阶段被确定为中间萌芽阶段（ 图2C）同的Silwet-77在用于第一浸渍0.05％，随后在第二次浸渍后期芽阶段（ 图2C）与0.03％略有减少的Silwet-77的浓度。转型也运作良好使用早期萌芽阶段（ 图2A）与低SILWET-77的浓度为0.003％，其次是第二浸渍与中间芽阶段（ 图2B）在较高的Silwet-77浓度为0.05％。 在这个协议中，一些其它参数进行尝试以优化转化率，但发现有对最终结果没有影响。实例包括浸渍，该植物放置在其侧面和盖在塑料从一天到两天后延伸的时间;使用超过1的OD为农杆菌培养，而不是0.5 - 1;增加浸渍时间为5 - 15分钟而不是1 - 2分钟。同样，我们还没有看到使用这些策略的转化率有什么影响。最有效的因素，但是，发现使用健康的植物在正确的花的阶段，以及使用的最佳的Silwet-77的浓度。我们注意到有两个浸渍时间间隔，工作不知何故比一次，即使一次浸渍也适用。 修改该协议可以通过reducin实现克的Silwet-77浓度低至0.003％，在第二次或第三次浸渍。自的Silwet-77是有毒的，在花过高的浓度导致显影不良，导致没有种子产量。浸渍频率可以减少到一个，与第二或第三事件消除，如果植物不看健康的芽没有发展良好。 这种技术的一个主要限制是由亚麻，从各花得到的种子的数量有限，和亚麻的长寿命周期产生花朵数量低。它采用6 - 8星期播种有准备的第一个浸渍的主要花蕾和一个额外的8 - 10周后倾去的T1代。总共，范围为5 - 需要6个月至获得T1代。不像其他的植物，开花随时随地的一年，一些亚麻品种花一年更好的在特定的时间。所以，周到的规划这项技术是非常重要的。 农杆菌 -经由花香浸介导的植物转化是亚麻变换适用而有效的方法，并可以用来代替以前使用的技术亚麻转化。花香浸法在本协议的修改将是适用于与任何其它植物物种的使用，而不是仅限于亚麻。

亚麻（ 亚麻 ）是广泛生长于它的纤维和油脂的重要作物。亚麻基因组的变换是可能的，如伤人， 农杆菌感染和共培养在组织培养中，施加后面再生生物射弹粒子或超声声处理技术。然而，这些技术具有许多缺点，包括倾向许多突变事件和一个延长的时间，以获得转基因株系。其中一些方法也可以是昂贵的并且需要熟练的和高效率的操纵器械的，导致低的恢复的幼苗。最重要的是，这些技术通常导致低转化速率2,6。 经由花香浸农杆菌介导的植物转化是一种简单而有效的方法，以产生转基因植物。它已被常规地并成功地用于许多植物物种，如拟南芥thalian1,4， 蒺藜 11，西红柿12，麦13和玉米10。然而，它并没有被认为是一种可行的技术亚麻变换由于多种因素，如低的数字鲜花由亚麻生产的，从各花，大种子大小，和厚涂层得到的种子的数量有限，这也可能是有问题的这种遗传转化的过程。此外，花浸技术的选择段需要发芽在含有抗生素，与转化的后代的植物介质转化的种子尊贵基于它们发芽并保持绿色的能力，同时非转化后代要么不发芽或发芽但漂白出迅速死亡。在目前的文献中，已经注意到，野生型亚麻趋于逃避高浓度的抗生素的选择，产生假阳性结果，并使得T1后代的选择基于对抗生素的耐药性更困难6,14。此外，当高浓度的抗生素的溶液中加入到选择培养基中，观察到的转化率显着下降9。 在这个协议中，我们使用的土壤杆菌和花香浸法来转换一个线路光纤亚麻，斯托蒙特卷云（响应和塑料），其已经显示出，通过改变其基因组3,5以响应于环境应力。为了克服抗生素逃逸的问题，我们已选择了做，而不是选择从T1叶的DNA的直接PCR检测，通过添加抗生素到植物的介质。我们采取了亚麻的简单解剖的优势，在追踪治疗时具体的花朵。这个跟踪系统允许选择来自特定的花朵和发芽的土壤种子无添加抗生素。阳性转化只需通过使用快速，高效的方法○叶检测DNA鉴定获得˚F直接PCR。我们的研究结果表明，花香浸法在这条线的亚麻工作得很好，并令人惊奇地导致了非常高的转化率（50％ - 60％），除前面对于拟南芥 ，这是据报道，观察到更高0.1 - 1 ％1，而且比其他植物物种10,12也较高。我们还测试了另一品种亚麻籽（胡麻）的，白求恩（稳定和非响应），以及我们的初步数据表明，花卉浸方法也适用于该品种的亚麻。 该协议的目的是要表明， 土壤杆菌和花浸可以用来产生转基因亚麻。我们表明，这种技术简单，价格低廉，而且比亚麻转化的其他方法更快。更重要的是它导致高得多的转化率比亚麻变换2,6的其他方法。 拟南芥的解剖结构，其中有许多树枝和鲜花，马KES难辨下跌和非蘸花对同一工厂。因此，大量的种子，每株大约20000的种子，需要进行筛选以鉴定阳性转化8。亚麻，另一方面，具有较少的分支（一个主支路和几个侧面分支）和更少的花，生产每植株约100粒种子，这使得有可能跟踪单个花朵和筛选过程中，选择特定的种子。 我们建议花香浸是将任何相关品种的亚麻，约200种的属适用的方法。此方法提供高得多的转化率比亚麻转化的其他方法。我们也提议的T1叶DNA的直接PCR筛选，就是要克服，往往会产生很多误报抗生素耐药性逃逸的问题的一个有效途径。直接PCR筛选可应用于任何其他植物物种，并且不限吨Ø亚麻。在这个协议中使用的简单的种子的跟踪技术可以应用到任何其他植物物种与分支解剖学类似于亚麻。

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flax seeds of the original Stormont Cirrus variety (PL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5&#34; pots</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potting soil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">greenhouse with appropriate light setting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose gel electropheresis equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">digital imaging setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silwet-77&#160;</td>
			<td>LEHLE SEEDS</td>
			<td>VIS-01</td>
			<td>Toxic, wear gloves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq Green Master Mix</td>
			<td>promega</td>
			<td>Part# 9PIM712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terra PCR Direct Polymerase Mix</td>
			<td>Clontech</td>
			<td align="right">639270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binary vector PRI 909</td>
			<td>Takara</td>
			<td align="right">3260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agrobacterium tumefaciens LBA4404 E</td>
			<td>Takara&#160;</td>
			<td align="right">9115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TOPO TA cloning kit</td>
			<td>invitrogen</td>
			<td>K4595-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sucrose&#160;</td>
			<td>fischer scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">electroporator and cuvettes</td>
			<td>bio-Rad</td>
			<td>165-2092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spinner&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">platic wrap and aluminum foil wrap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">speedSTAR DNA polymerase</td>
			<td>Takara</td>
			<td>RR070A/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QlAquick gel extraction kit&#160;</td>
			<td>Qiagen</td>
			<td align="right">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAGEN plasmid mini kit</td>
			<td>Qiagen</td>
			<td align="right">12123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SalI-HF enzyme</td>
			<td>NEB</td>
			<td>R3138S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SacI-HF enzyme</td>
			<td>NEB</td>
			<td>R3156S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA ligation kit</td>
			<td>NEB</td>
			<td>M0202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murashige Skoog</td>
			<td>sigma</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>fisher scintific</td>
			<td>A360-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

floral-dip transformation of flax (linum usitatissimum) to generate transgenic progenies with a high transformation rate

经由花香浸农杆菌介导的植物转化是在植物转化领域的广泛使用的技术，并已报道成功用于许多植物物种。然而，亚麻（ 亚麻 ）由花香浸变换尚未见报道。这个协议的目标是建立该农杆菌和花香浸方法可用于产生转基因亚麻。我们表明，这种技术是简单，廉价，​​高效，而且更重要的是，给出了一个较高的转化率比亚麻变换的当前可用的方法。 总之，亚麻花序浸入农杆菌溶液携带二元载体的质粒（T-DNA片段加上亚麻插入序列，LIS-1）1 - 2分钟。这些植物被平放在自己身边24小时。然后，将植物保持在正常生长条件下，直到下一个处理。进程列表浸渍第重复2 - 3倍，约10 - 浸渍在14天的时间间隔。所述T1种子收集发芽的土壤。经过大约两个星期，后代处理通过直接PCR检测; 2 - 3叶每株加上适当的T-DNA引物使用。阳性选择转化体并生长至成熟。转化率是意想不到的高，用50 - 从处理的植物为阳性转化的种子的60％。这是一个较高的转化率比那些报道的拟南芥和其他植物物种，使用的花香浸改造。它也是最高的，这已被报道，到目前为止，对于亚麻变换使用其他方法进行转化。

图1 -图4示出的一些协议中的步骤。在图1和2，围绕花序芽的叶子切成它们暴露在农杆菌细胞以及用于开发协议不同芽阶段; 图3示出了亚麻花香浸的过程。 图4，示出了例的如何主要和侧枝可以标记和花如何个体可以跟踪和识别。 图5示出的T1后代可以如何发芽所述MS植物介质，然后移植到土壤中的成熟度。 图6示出了如何野生类型亚麻竟能高浓度卡那霉素，文献6,9,14证实先前的调查结果。 图7示出的直接PCR扩增从阳性的T1转化体的一个例子。在T1鲜花从m收集单T0植物艾因和方竹笋。如可以从该直接PCR中可以看出，8/12的T1植物测试了经PCR阳性和已经扩增跨越在T-DNA的不同区域。我们的引物也是LIS-1插入物和多克隆位点（ 图7B和C）之间而设计的。我们使用的额外的引物从植物二元载体以扩增在T-DNA的不同段，诸如左边框和NOS终止子（数据未示出）或右边界和多克隆位点（ 图7D）。具体到LIS-1的插入引物也用在这个协议中（数据未示出）。在表1中提供的引物列表。然而，这些引物的序列依赖于T-DNA的植物二元载体的序列和用于花卉浸插入。我们也注意到，有从主侧枝收集花之间的转化率没有显著差异。 图1.切割各地的主要花序芽的叶子给他们暴露在Agrobacterim细胞（A）的芽被落叶覆盖。（B）叶已被切断周围的芽揭露他们。（C）从植物放大图像在（A）切割，露出芽后。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52189/52189fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52189/52189fig2highres.jpg" /> 图2中使用了这个协议，确定要使用的浸花的最佳舞台不同芽阶段。（A）的早期萌芽是approxima<html> tely为2 mm。（B）的中期阶段芽约为5毫米（C）后期芽约为1厘米左右。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52189/52189fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52189/52189fig3highres.jpg" /> 图3.亚麻花香浸渍的方法。 （A）中的主花序浸在含有农杆菌细胞的浸润介质。（B）从（A）的放大。（C）中浸渍植物平放直到第二天，和浸渍分支覆盖有塑料保持高湿度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52189/52189fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> jove_content"FO:保持together.within页="总是"&gt; <img alt="图4" src="/files/ftp_upload/52189/52189fig4highres.jpg" /> 图4.花的跟踪和种子收集的过程中，从T0处理厂（一）全厂的主要分支（在中心最高的分支）的一个例子和侧分支（B - D）。一个。例如，从不同的分支花（E）从单花收集的种子的例子（标有- K）。从主枝<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52189/52189fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52189/52189fig5highres.jpg" /> 图5. T1幼苗生长过程中没有抗生素的选择。（A）T1种子发芽的植物MS培养基。（B）正转化，通过直接PCR检测，被移植到土壤和发育成熟。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52189/52189fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52189/52189fig6highres.jpg" /> 图6.抗生素逃生，为T1的选择问题，通过直接PCR筛选克服。（A）在发芽的植物MS培养基无抗生素野生型亚麻种子。（B）野生型亚麻种子发芽的植物MS +介质浓度的增加的卡那霉素（200微克/毫升，600微克/毫升，1毫克/毫升）。（C）的野生型亚麻和T1上萌发的MS植物培养基用2毫克/毫升卡那霉素。（D）中的PCR野生型亚麻种子用卡那霉素引苗，全部AMPL后指定的卡那霉素基因（传说:EZ1:DNA标记，FlaxS，野生型flaxS，C:控制非浸支，麻:从花T1后代"是"从主分支收集，SC，SD，SE，SF:不同的花"C，D，E，F"从侧面分支，W收集T1后代:无DNA） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52189/52189fig6large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52189/52189fig7highres.jpg" /> 图7.使用直接PCR方法T1后代成功的PCR扩增的一个例子。植物二元矢量+克隆LIS-1插入（A）图。蓝色箭头指示的直接PCR筛选（从宝修改）用PCR引物的位置。（B）PCR引物M13F + 3"（C）PCR引物M13R + 18A <stro纳克&gt;（D）中的PCR用引物右边界（RB）和多克隆位点（MCS）**每个泳道表示来自从C收集的个人花T1:控制分支（非浸渍）中，M:主枝花AG，S:侧枝花BC&#39;。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52189/52189fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>正向引物的序列来源</td><td>序列5&#39;-3&#39; </td><td>反向引物序列源</td><td>序列5&#39;-3&#39; </td><td>退火温度图（°C） </td><td>延长时间（秒） </td><td>预期大小（bp）的</td></tr><tr><td> M13F（T-DNA） </td><td> CTGCAAGGCG ATTAAGTTGG </td><td> 3&#39;（LIS-1插入） </td><td> GAGGATGGAA GATGAAGAAGG </td><td> 57 </td><td> 40 </td><td> 450 </td></tr><tr><td> 18A（LIS-1插入） </td><td> TATTTTAACCC <td> M13R（T-DNA） </td><td> ATTAGGCACC CCAGGCTTTA </td><td> 57 </td><td> 40 </td><td> 520 </td></tr><tr><td> MCS（T-DNA） </td><td> TGGTCATAGC TGTTTCCTGTG </td><td> RB（T-DNA） </td><td> TTTAAACTGA AGGCGGGAAA </td><td> 60 </td><td> 20 </td><td> 200 </td></tr><tr><td> LB（T-DNA） </td><td> TTTGATGGTG GTTCCGAAAT </td><td> NOS（T-DNA） </td><td> GAATCCTGTT GCCGGTCTT </td><td> 60 </td><td>三十</td><td> 380 </td></tr><tr><td> NPTII（T-DNA） </td><td> GCGATACCGT AAAGCACGAG </td><td> NTPII（T-DNA） </td><td> GCTCGACGTT GTCACTGAAG </td><td> 65 </td><td> 45 </td><td> 502 </td></tr></tbody></table> 表1.一些用于直接PCR检测的引物。

Watch this Scientific Journal Video about Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate at JoVE.com

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

在这里，我们提出了一个协议，通过花卉浸利用农杆菌介导的植物转化改造亚麻。该协议是简单的执行，价格便宜，但得到更高的转化率比亚麻改造当前可用的方法。

亚麻花香浸改造（<em&gt;亚麻</em&gt;）生成的转基因后代具有高转化率

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

floral-dip-transformation-flax-linum-usitatissimum-to-generate

Nanyang Technological University

Research

JoVE Journal

Biology

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

29.0K 次观看.  Case Western Reserve University. 在这里，我们提出了一个协议，通过花卉浸利用农杆菌介导的植物转化改造亚麻。该协议是简单的执行，价格便宜，但得到更高的转化率比亚麻改造当前可用的方法。 

视频: Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

科研

生物学

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

高效的聚乙二醇（PEG）介导的转化苔小立碗藓</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

农业细菌-中生菌遗传转化、转基因生产及其在水稻雄性生殖发育研究中的应用

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

多活性猪猪Bac转座酶介导的秋粘虫，蜉蝣属果蝇蛆中的种系转化

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

丝状蓝藻腔隙的自然转化、蛋白表达和低温保存

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...