这项研究是由来自全球CLL研究基金会的资助。得克萨斯大学MD安德森癌症中心的大学是由美国国立卫生研究院的部分通过一个癌症中心支援津贴（P30CA16672）的支持。

1.识别微小RNA基因的启动子转录因子结合位使用数据挖掘方法<ol><li>使用美国加州大学圣克鲁兹分校（UCSC）基因组浏览器作为提取DNA元素（ENCODE）项目的百科全书的一部分生成染色质免疫沉淀（芯片）测序数据。 <ol><li>打开在UCSC基因组浏览器表浏览器。 </li><li>使用以下规格提取表:类群:（哺乳动物），基因组（人），大会:（Feb2009（GRch37 / hg18）），组（监管），跟踪（TxnFactorChIP），表（weEncoderesTFbsCo7steredv3），区域（基因组），输出格式（从选定表中的所有基因）。 </li><li>从保存1.1.2输出为.txt文件，并到电子表格中。 </li><li>排序和过滤感兴趣的转录因子（ 例如 ，STAT2）。 </li></ol></li><li>使用基于H3K4me3的表观遗传签名的microRNA发起人提供Baeer 等人名单。9 </suP&gt;。这个列表复制到一个文本文件。 </li><li>根据人类基因组坐标相匹配时，数据从1.1和1.2（ 例如 ，STAT3上推定的微小RNA的启动子结合）映射，并使用C语言编写的尖锐的代码确定为在补充所述编码文件1中位结合亲和力。 </li></ol> 2.使用的shRNA下调利率的转录因子的表达<ol><li>板从于10cm板293人胚肾细胞系在约50％汇合（含10％FBS）的1.5×10 6个细胞。 </li><li>从用含有shRNA的绿色荧光蛋白（GFP）的慢病毒的5微克293人胚肾细胞系转染细胞定向到感兴趣的转录因子，并与使用转染试剂，根据制造商的协议的贴壁细胞的包装载体的5微克。 </li><li>作为对照，转染从293人胚肾细胞系的细胞加扰的shRNA和根据制造商的协议的包装载体。 </li><li>保持对细胞转染混合物16小时（在37℃，CO 2培养箱），然后改变媒体至10ml新鲜的10％DMEM培养基含10％FBS。 </li><li>等待48小时转染后，离心细胞培养物（300 XG，5分钟），并收集感染上清。通过0.45μm注射器式滤器（25毫米无表面活性剂的醋酸纤维素膜）过滤上清液以除去任何漂浮的细胞。 </li><li>浓缩上清液，用超离心过滤装置与100 kDa的门槛收集慢病毒。旋转950 XG 30 - 60分钟，直到量一直精矿小于250微升。在-80℃保存浓缩病毒。 </li><li>转染用慢病毒的细胞。从-80℃冷冻机中取出冷冻的慢病毒和解冻至室温。转移100μl病毒上清到一个新的1.5 ml离心管。 <ol><li>布林克音量在管到1ml具有降低的血清培养基。聚凝胺添加到1ml病毒悬浮液为10ng / ml的终浓度，轻轻混合并让该混合物静置5分钟。 </li></ol></li><li>离心机5×10 6个细胞的每个转导并轻轻重悬细胞沉淀在0.5ml的含有病毒的介质。让细胞留在培养箱中4 - 24小时，然后加入0.5毫升20％FBS的培养基中，以10％的FBS的最终浓度。 </li><li>等待48至72小时，并弄脏用碘化丙锭（PI），并根据制造商的说明绿色荧光蛋白（GFP）的细胞。避光细胞，并用流式细胞分选仪来测量GFP + / PI的价格-细胞。由于PI污渍只有死细胞，这一比率在活细胞的转染效率的估计。 </li><li>如前面所述10排序的阳性细胞数来GFP表达（GFP +）通过FACS分拣机。 </li><li>利用Weste氡免疫印迹如前所述10之前，并与指定的shRNA感染感兴趣的细胞（例如，白血病细胞）后，以确定所关注的转录因子的水平。 </li></ol> 3.确定微RNA转录的表达水平与转录因子的shRNA转染细胞<ol><li>使用商业试剂盒根据制造商的协议的RNA分离。 </li><li>标记的RNA和其杂交到微RNA微阵列11。 </li><li>确定与转录因子的shRNA或空载体对照5转染的细胞中差异表达的microRNA。 </li><li>验证用于使用实时PCR 5的最差异表达的微小RNA的微阵列结果。 </li></ol> 4.确定生物信息学和shRNA的途径之间的重叠在描述转录因子相关转录<ol><li>要DET貂窝藏转录因子结合在其发起人地点和转录因子的shRNA表达下调转染细胞的microRNA基因的预期和观察到的比例，做到以下几点: <ol><li>获得窝藏感兴趣的转录因子在从1.3生成的列表中的启动子/总微小RNA基因的比率。这个名单是预期的比率（ 例如 ，微小RNA基因用STAT3结合位点/总微小RNA基因测试= 0.25）。 </li><li>确定在转录因子的shRNA转染的细胞下调从3.3生成的列表并且具有从1.3生成的列表兴趣结合位点的转录因子的基因的数目。下调的基因的这个数/总数是所观察到的比例（ 例如 ，微小RNA基因用STAT3结合位点/总数下调微小RNA = 0.6）。 </li></ol></LI&gt; <li>使用χ2统计比较上述生成的观察值与预期的比率，并确定是否在转录因子的shRNA转染的细胞下调的基因的列表与在其启动子的转录因子结合位点富集。 </li></ol>

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The authors declare no competing financial interests.

蛋白质编码基因的RNA聚合酶II-依赖性转录背后的机制已被广泛研究。而这些元素组成只有1％ -人类基因组的2％，从ENCODE项目证据表明，人类基因组的80％以上的可经历转录17和什么调节非编码DNA元件的转录在很大程度上仍然未知6 。 几项研究，表明聚合酶II也负责的一些非编码蛋 ​​白的基因，包括微RNA 6的转录，导致我们开发出从公共可用的资源，?...

微RNA是内生的小非编码调控RNA，通常在转录后水平起作用的mRNA表达的作为负调节。大约1000非编码20至25个核苷酸长的微小RNA在人类基因组中的1,2-找到。微RNA调节通过微RNA及其互补种子匹配序列，其通常位于3&#39;非翻译区的靶mRNA的（UTR）的种子序列之间规范碱基配对的基因表达。总的来说，微RNA调控的蛋白质编码基因3超过30％，但仅知之甚少从微RNA的DNA的转录。它已被提出，微RNA转录的调节类似于表达4,5。特别是，类似于其在促进蛋白质编码基因的转录活性，转录因子被认为激活微RNA 6的转录。转录因子-MicroRNA相互作用已被报告为基因表达7的调制因子，并且还可以形成反馈和前馈回路。例如，Yamakuchi 等报道的反馈环路，其中的p53诱导microRNA34a，表达这反过来又抑制了p53的阻遏SIRT的翻译，并由此提高p53活性8。 而微RNA的转录因子依赖性表达的具体例子，已报道，缺乏其中提供了关于感兴趣的转录因子如何调节微小RNA-转录的表达信息的被接受的方法。本文所提出的协议的目的是提供微小RNA-转录的转录依赖性因子调控的进行了深入的说明。通过结合公开可用的数据，生物信息学工具，并使用微阵列技术，谁遵循这个算法的研究人员将能够捕获基因组规模如何的任何在任何感兴趣的细胞类型的转录因子调节微小RNA-转录的表达和在调节微RNA表达探索转录因子mRNA的推定贡献。

<table border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Lipofectamin 2000</td>
			<td style="width:204px;">Life Technologies</td>
			<td style="width:119px;">11668027</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 um syringe filter&#160;</td>
			<td style="width:204px;">Thermo Scientific (Nalgene)</td>
			<td>190-2545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon ultracentrifugal filter device with threshold of 100kDa&#160;</td>
			<td style="width:204px;">Merck Millipore</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polybrene&#160;</td>
			<td style="width:204px;">Merck Millipore</td>
			<td style="width:119px;">TR-1003-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol reagent&#160;</td>
			<td style="width:204px;">Life Tachnologies (Invitrogen)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">293 Cell line human</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:119px;">85120602</td>
			<td></td>
		</tr>
	</tbody>
</table>

describing a transcription factor dependent regulation of the microrna transcriptome

而蛋白质编码基因的转录调控被广泛研究，知之甚少转录因子如何参与非编码RNA的转录，特别的微RNA。在这里，我们提出了一个战略，研究使用规范公开提供的数据，计算资源和高吞吐量数据的microRNA的转录转录因子的潜在作用。我们使用的H3K4me3的后生签名从ENCODE项目识别微RNA启动子和染色质免疫沉淀（ChIP）-sequencing数据，以确定被富含转录因子结合位点的微RNA启动子。通过转染与shRNA的靶向感兴趣的转录因子和使细胞微RNA阵列感兴趣的细胞中，我们研究这转录因子对微RNA转录的影响。作为一个说明性的例子，我们使用我们的研究STAT3的慢性Ly的微小RNA转录的影响mphocytic性白血病（CLL）细胞。

STAT3是其通常导致具有抗凋亡和增殖作用12个基因的转录的转录因子。无论是STAT3也影响了非编码RNA的转录，目前尚不得而知。在所有CLL细胞STAT3是丝氨酸残基707组成10,13磷酸化。磷酸STAT3梭到细胞核，与DNA结合，并激活已知由酪氨酸pSTAT3的在其他细胞类型10激活的基因。因为CLL特征是微小RNA网络14的全球解除管制，我们推测，丝氨酸pSTAT3的的...

Watch this Scientific Journal Video about Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome at JoVE.com

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

描述了微RNA转录的转录因子依赖调节

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

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8.3K 次观看.  Rabin Medical Center. Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest. 

视频: Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

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生物工程

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.

监视依赖黄素单加氧酶和氧化还原半反应，用停流分光光度法

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

一个<em&gt;在体外</em&gt;酶测定来测量转录抑制作用镓（Ⅲ）和H<sub&gt; 3</sub&gt; -5,10,15-三（五氟苯基）corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...