我们感谢春新王建设pTSO2：：GUS和图1，德特勒夫威格尔的照片分享有益的意见的GUS万无一失的协议，帕哈Sijacic和考特尼荷伦德。 Z. L S实验室的研究是由美国国家科学基金会（IOB0616096和MCB0744752）的支持。 ZL是由美国马里兰大学农业试验站的部分支持。

一，花香浸拟南芥的转化<ol><li>改造前约1个月，大约有10-15％的盆栽植物拟南芥种子播撒到4至8盆（5平方英寸的花盆）。与正常生育的植物，4盆植物每质粒的构建就足够了。如果一个人改造拟南芥突变生育率下降，增加相应的盆数。 </li><li>植物生长标准条件下（16小时光照，8小时黑暗，在22 ° C）。如果较短的一天或更低的温度下生长的植物，他们将需要更长的时间，花和更长的改造做好准备。良好的植物护理，确保健康的植物，这是成功转型的关键。 </li><li>当植株只是狂奔，并开始开花。他们现在准备转型。为了提高转换效率，改造前的3-4天，剪掉主花序芽，尽快为他们狂奔，以鼓励更多中学拍摄形成。植物浇水改造前的一天。这确保土壤不会吸收太多渗透媒体在花香浸。 </li><li>改造前两天，接种40毫升含有适当的抗生素的LB农杆菌菌株GV3101中携带的pTSO2（pTSO2情况下的 GUS，50微克/ ml卡那霉素：）：：GUS的构造。 pTSO2：：GUS在pBIN20载体，拥有NPTII基因，赋予抗卡那霉素3克隆。在摇床过夜增长在28-30 ° C。 </li><li>翌日，转移到含有50微克/ ml卡那霉素LB400毫升8毫升过夜培养。在摇床过夜增长在28-30 ° C。 </li><li>在转型的一天，当外径 600农杆菌文化达到约0.8（ 外径 600 0.5 TO1之间是可以接受的），降速在8分钟在5000RPM农杆菌过夜培养。 </li><li>在1升悬浮农杆菌渗透介质颗粒（升）（10MM氯化镁2，5％的蔗糖，0.44mM 6 benzyladenine（BA），0.3％silwet L - 77，1X Gamborg的维生素解决方案和灭菌水）。媒介并不需要进行高压灭菌，但需要作出新鲜。成菜（如8“X15”X2“硼硅玻璃盘），倒入1升含农杆菌渗透介质。 </li><li>反转锅（植物不会脱落）和渗透液浸所有的植物地上部分，一锅煮5分钟举行一次。虽然植物的地上部分是渗透液中浸泡，避免让土壤浸泡渗透媒体，以减少对土壤真菌生长的机会。 </li><li>浸渍后，放在几层纸巾蘸盆就在自己身边，在一个托盘。纸巾浸泡渗透媒体的访问量。封面用保鲜膜，以确保高湿度的托盘。放置到植物生长室的托盘。 </li><li>翌日，除去保鲜膜和纸巾，和地方盆直立。不要将这些植物的水为四，五个多天。之后，维持植物正常，直到他们种子，并收集散装种子。 </li><li> MS培养基中含有50微克/ ml卡那霉素（重量2.2克Murashige和Skoog不含维生素基底盐倒入板（150x15mm培养皿），1M氢氧化钠溶解在500毫升的水，pH值至5.8，加4革兰氏琼脂，高压灭菌，冷却和添加卡那霉素50μg/ ml的终浓度）。板都保持在4℃直至需要。 </li><li>要选择抗生素耐药性的转基因植物，需要至少20000种子屏幕。 0.1克约5000种子。重量和估计的金额的种子。 </li><li>他们用70％乙醇30秒15毫升猎鹰管清洗消毒种子。用无菌水冲洗种子一次。包含商店买来的，5％漂白水（Clorox公司）1:10稀释的溶液中浸泡30秒的种子。无菌水冲洗3至6倍的种子。在无菌罩，吸管约5000到每个MS板（卡那霉素）的种子，传播的种子均匀板，微孔3M胶带密封板，以避免污染。 </li><li>孵育板在4 ° C在3-4天的黑暗，然后转板一腔（可能是相同的植物生长室）。经过约14天，转基因苗保持绿色，但非转基因​​的脸色苍白，奄奄一息。放缓拉根从中长期放置在土壤中，转基因苗转移到土壤。 </li></ol>二。检查pTSO2：：GUS表达模式<ol><li>收获pTSO2：：GUS转基因苗，他们在寒冷的90％丙酮在玻璃闪烁瓶或离心管保存在冰上。聚苯乙烯微孔板（聚丙烯），也可以使用，如果有大量的样品进行分析。 </li><li>所有样品都收获后，在室温下放置20分钟的小瓶。在这段时间内没有X - Gluc（0.2％Triton X - 100，50mm的NaHPO PH7.2缓冲4，2MM亚铁氰化钾，2MM钾铁氰化钾），并置于冰上新鲜的染色缓冲。 </li><li>从样本中删除丙酮和添加染色缓冲。 </li><li> X - Gluc染色的溶液（0.2％的Triton X - 100，4 NaHPO 50mm的缓冲液PH7.2，2MM钾亚铁氰化物，2mm的铁氰化钾，采用2mm x - gluc） 。从样品中取出染色缓冲和添加样品含有X - Gluc染色缓冲。 </li><li>下15至20分钟的真空渗透的样品。慢慢松开真空和验证，所有样品染色溶液的表面之下下沉。如有必要，重复，直到所有样本下沉渗透，被释放后的真空。 </li><li>孵育在37 ° C与X - Gluc染色缓冲过夜。 </li><li>从孵化器中取出样品取出染色缓冲。在连续乙醇系列清洗样品在室温（20％，35％和50％的乙醇）改变每次30分钟。 </li><li>在FAA固定液（50％乙醇，5％的甲醛，10％的醋酸，其余水）为30分钟，在室温下的组织修复。删除美国联邦航空局（FAA）​​和添加70％的乙醇。此时组织可配备一台数码相机在解剖显微镜下可视化和拍照。深蓝色的颜色表示 TSO2是表示（图1） 。另外，组织可存放在4 ° C以供日后观赏。 <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="图1" /> 图1 pTSO2：：GUS 基因在转基因苗（一），（二）侧根，并根尖（C）。请注意，TSO2发起人是非常年轻和分裂组织中的积极。 （C）所示的零星的表达模式是在特定的细胞周期基因表达的典型。 </li></ol>代表性的成果如果做得正确，转化效率约为0.1-0.2％。换句话说，应该得到筛查每5000种子5-10转基因苗。平均，约100个​​转基因株系可转化野生型植株的4盆。 对于转基因苗含有pTSO2：：GUS基因构建3，深蓝色的色调反映了GUS活性，是在积极的分裂中的细胞，包括嫩叶，茎尖，根尖和侧根原基（图1）。非均匀的零星模式的特点是细胞周期阶段的具体体现。

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

转化效率是由许多不同的因素，下面讨论的： <ol><li>植物的健康和他们的年龄是最重要的。约一个月的老厂生产大量未成熟的花芽，是理想的。修剪主芽，以鼓励中学改造前3-4天拍摄形成会增加转化效率。老厂将产生转化，但在较低的利率。 </li><li>生育的植物是重要的。如果一个人改造与生育率下降的突变体，植物将需要更多的浸渍。 </li><li> 0.3％Silwet L - 77渗透缓冲区中的表面活性剂被认为?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>6-benzyladenine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>852430</td>
<td>&nbsp;</td>
<tr>
<td>Silwet-77</td>
<td>&nbsp;</td>
<td>Crompton Corp.</td>
<td>&nbsp;</td>
<td>Toxic, wear gloves</td>
</tr>
<tr>
<td>Murashige and Skoog Basal medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>Gamborg's vitamin solution 1000X</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1019</td>
<td>&nbsp;</td>
<tr>
<td>X-Gluc (5-bromo-4-chloro-3-indolyl &beta;-D-glucuronide cyclohexamine salt</td>
<td>&nbsp;</td>
<td>Biosynth</td>
<td>B-7300</td>
<td>Toxic, light sensitive keep in the dark</td>
</tr>
<tr>
<td>Micropore 3M Tape</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19027761</td>
<td>&nbsp;</td>		</tbody>
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floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

一个有机体，引入外源基因的能力，是现代生物学和生物技术的基础。在该模型中的开花植物<em&gt;拟南芥</em花浸变换方法<sup&gt; 1-2</sup&gt;已取代所有以前的方法，因为它的简单，效率，成本低。具体来说，年轻开花的芽<em&gt;拟南芥</em&gt;植物的溶液浸泡<em&gt;农杆菌</em&gt;携带特定的质粒结构。浸渍后，植物恢复到正常的生长和产量的种子，很小的比例，其中与外源基因的转化，可以选择含抗生素的培养基上。这花香浸方法，大大地促进<em&gt;拟南芥</em&gt;研究大大促进了我国植物基因功能的理解。在这项研究中，我们使用花香浸法记者基因改造，<em&gt;β-葡萄糖醛酸酶（GUS）的</em&gt;下，控制<em&gt; TSO2</em&gt;推动者。<em&gt; TSO2</em&gt;，核苷酸还原酶（RNR）小亚基编码<sup&gt; 3</sup&gt;，是一个细胞周期调控基因在细胞周期S期dNDP合成的必要条件。审核<em&gt; GUS</em&gt;在转基因的表达<em&gt;拟南芥</em&gt;苗显示，<em&gt; TSO2</em&gt;表示积极分裂组织。报告的实验方法和材料可以很容易地适应不仅为研究，而且还对教育，在高中和大学水平。

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

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

本文介绍了花卉浸方法<em&gt;农杆菌</em&gt;介导的转化<em&gt;拟南芥</em&gt;。通过引入一个细胞周期调节的发起人，记者，<em&gt; pTSO2：β-葡萄糖醛酸酶（GUS）的</em&gt;，到<em&gt;拟南芥</em&gt;，我们演示了如何检测到GUS基因在转基因苗记者表达。

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 ...

floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2

Research

JoVE Journal

Biology

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

35.8K 次观看.  University of Maryland College Park. 本文介绍了花卉浸方法农杆菌介导的转化拟南芥。通过引入一个细胞周期调节的发起人，记者， pTSO2：β-葡萄糖醛酸酶（GUS）的，到拟南芥，我们演示了如何检测到GUS基因在转基因苗记者表达。

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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

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

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

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 ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...