1）细胞制备<ol><li>当使用贴壁细胞，它是必要trypsinize和收集细胞前电的。 </li><li>为了比较各转染小鼠胚胎成纤维细胞（MEF）的文化，代表三种不同的通道号码，，在每个烧瓶中执行以下。 </li><li>吸脱落细胞培养基。 </li><li>添加PBS洗细胞。 </li><li>删除PBS，补充足够的胰蛋白酶盖细胞，并等待几分钟，使胰蛋白酶来分离细胞。 </li><li>用显微镜检查验证条件下的细胞培养瓶，嫌烧瓶中分离细胞，然后再次检查烧瓶，确保细胞都分离。 </li><li>等待更多的时间和必要时重复。 </li><li>所有的细胞中分离后，加入含药血清的介质中和胰蛋白酶。 </li><li>细胞转移到离心管中，离心沉淀细胞（RCF = 300 XG）。 </li><li>删除已知体积的PBS上清和悬浮细胞。 </li><li>计数细胞。 </li><li>转移到一个新的管适当体积的细胞悬液，以提供所需数量的细胞进行实验（您将需要在1 × 10 6细胞/ ml密度细胞每孔150μL）。 </li><li>离心机细胞。 </li><li>取出上清和悬浮细胞在基因脉冲电缓冲区实现了1 × 10 6细胞/ ml的细胞密度适当的音量。 </li><li>加入质粒的细胞悬液，每毫升20微克，轻轻混匀。 </li></ol> 2）电穿孔容器的设置和电板的设置和电<ol><li>插入基因的脉冲MXcell电系统的电源模块板腔。 </li><li>移液器150μL细胞的混合物或缓冲到一个96孔的电板井。 </li><li>板腔和脉冲沽盘。 </li><li>从室取出钢板。 </li><li>吹打在每口井的向上和向下混合以及内容。 </li><li>从每口井预热的缓冲液在12孔板的细胞转移。 </li><li>塔盘分发细胞和它放在孵化器。 </li><li>让细胞恢复为24小时。 </li></ol>试管设置和电<ol start="9"><li>拔下电源模块板的基因脉冲MXcell电系统和插件在ShockPod™比色皿室室。 </li><li>吸取600μL的细胞悬液到0.4厘米的空隙电比色皿。 </li><li>比色皿在ShockPod室，并提供电脉冲。 </li><li>从室中取出比色皿。 </li><li>吹打在比色皿的向上和向下混合比色皿的内容。 </li><li>从每口井预热的缓冲液在12孔板的细胞转移。 </li><li>塔盘分发细胞和它放在孵化器。 </li><li>让细胞恢复为24小时。 </li></ol> 3）代表性的成果在转染细胞，并允许他们重新后，定性分析转染效率，使用啶显微镜，并定量，用流式细胞仪检测。 <img src="/files/ftp_upload/1662/1662fig1.jpg" alt="图1" /> 图1。已成功电穿孔和现在的绿色荧光蛋白基因表达的细胞出现啶显微镜下。 <img src="/files/ftp_upload/1662/1662fig2.jpg" alt="图2" /> 图2。查看相衬下的细胞，使转染和未转染细胞的可视化。这些细胞暴露在200V的最低电压电脉冲。细胞主要是由于高密度融合。 <img src="/files/ftp_upload/1662/1662fig3.jpg" alt="图3" /> 图3。epifluorescence下的同一领域的细胞表达绿色荧光蛋白标记的数量，但这些都只是一个很小的比例在前面的图像中可见的细胞。 <img src="/files/ftp_upload/1662/1662fig4.jpg" alt="图4" /> 图4。250V，相衬下看到活细胞总数略有下降。 <img src="/files/ftp_upload/1662/1662fig5.jpg" alt="图5" /> 在图5。epifluorescence，人们可以看到，绿色荧光蛋白表达细胞的数量增加。 <img src="/files/ftp_upload/1662/1662fig6.jpg" alt="图6" /> 图6的最高电压。应用，375V，有明显的活细胞较少。 <img src="/files/ftp_upload/1662/1662fig7.jpg" alt="图7" /> 图7。然而，剩下的细胞的很大比例是表达GFP 。取决于哪些条件是最佳的实验设计。在一些实验中的转染的细胞数量最多的可能是最优的，在其他实验的比例最高转可能是最好的。 我们是在细胞，GFP阳性每个条件下，如何的百分比与细胞年龄的不同而有所差异的百分比感兴趣。流式细胞仪可以提供有关每一个不同的电击条件下转染结果的定量信息。 <img src="/files/ftp_upload/1662/1662fig8.jpg" alt="图8" /> 图8：这里的细胞，绿色荧光蛋白在5 12电条件下的每个细胞都表明通过积极的百分比。最大的转染百分率约80％的测试电压最高的指数衰减脉冲，条件6，和70％下最强的方波脉冲下，条件12。 <img src="/files/ftp_upload/1662/1662fig9.jpg" alt="图9" /> 图9。转百分比的总体格局与细胞通过电穿孔前的9倍，几乎是相同的的，但在转染的百分比略有下降。 <img src="/files/ftp_upload/1662/1662fig10.jpg" alt="图10" /> 图10这里显示的是在通过显示一个相对年轻的细胞在转染的百分比显着下降的13个细胞，这些细胞的GFP细胞的百分比。转百分比最高约一半证明后尽快隔离尽可能使用健康细胞的重要性与年轻细胞来实现的。 <table border="1"><tbody><tr><td colspan="3">用于转染MEF的使用基因脉冲MXcell细胞的电穿孔条件</td></tr><tr><td>条件（1-6） 指数衰减脉冲， 所有350用友，1000ohm </td><td>电压（V） </td><td></td></tr><tr><td> 1 </td><td> 200 </td><td></td></tr><tr><td> 2 </td><td> 250 </td><td></td></tr><tr><td> 3 </td><td> 300 </td><td></td></tr><tr><td> 4 </td><td> 326 </td><td></td></tr><tr><td> 5 </td><td> 350 </td><td></td></tr><tr><td> 6 </td><td> 376 </td><td></td></tr><tr><td>条件（7-12） 方波脉冲， 所有与2000年用友，1000欧姆，和1个脉冲</td><td>电压（V） </td><td>脉冲持续时间（毫秒） </td></tr><tr><td> 7 </td><td> 200 </td><td> 10 </td></tr><tr><td> 8 </td><td> 250 </td><td> 10 </td></tr><tr><td> 9 </td><td> 300 </td><td> 10 </td></tr><tr><td> 10 </td><td> 200 </td><td> 20 </td></tr><tr><td> 11 </td><td> 250 </td><td> 20 </td></tr><tr><td> 12 </td><td> 300 </td><td> 20 </td></tr></tbody></table>

作者采用Bio - Rad公司实验室产生本文中使用的试剂和仪器

此视频文章演示了如何使用MXcell电系统可以轻松地找到MEFs或其他主要细胞株的最佳电条件。 96孔板格式允许对许多重复实验或优化的条件下同时进行，这可以消除许多不同的实验需要。在进行此过程中，人们应该记住使用健康细胞后尽快隔离，尽可能使用电条件，匹配电缓冲区。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gene Pulser&reg; Electroporation Buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2676</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; Electroporation System</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2670</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; ShockPod&trade; Cuvette Chamber</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2673</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; Electroporation System With ShockPod&trade; Cuvette Chamber</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2674</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

using the gene pulser mxcell electroporation system to transfect primary cells with high efficiency

这是越来越明显，电是最有效的方法，引入的原代细胞的质粒DNA或siRNA。基因脉冲MXcell电系统和基因脉冲电击缓冲液（BIO - RAD）专门开发的，很容易转染哺乳动物细胞和难以转染的细胞，如小学和干细胞，的核酸。我们将演示如何执行一个简单的实验，以迅速确定最佳电条件。我们将演示如何通过电条件的范围，这样一个实验可以在同一时间进行优化执行运行中的几个样本。我们还将展示最佳条件，确定如何使用96孔电板可以使用标准电试管，促进电板开关，电试管，同时保持相同的电效率。在录像中，我们还将讨论一些可能导致电击实验的成功或失败的的关键因素。

Watch this Scientific Journal Video about Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency at JoVE.com

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

此过程说明如何使用基因脉冲MXcell电系统，以迅速和容易地识别小鼠胚胎成纤维细胞（MEFs）或其他主要细胞的最佳电条件。故障排除注意事项还讨论了相关的视频。

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

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

using-gene-pulser-mxcell-electroporation-system-to-transfect-primary

Research

JoVE Journal

Biology

24.1K 次观看.  Bio-Rad Laboratories, Inc.. 此过程说明如何使用基因脉冲MXcell电系统，以迅速和容易地识别小鼠胚胎成纤维细胞（MEFs）或其他主要细胞的最佳电条件。故障排除注意事项还讨论了相关的视频。

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

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

电穿孔分枝杆菌

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

科研

生物学

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

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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 ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

反向遗传吗啉使用方法心脏心室射出转染多难靶组织中的斑马鱼幼虫

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...