第1步： 解冻293细胞的慢病毒生产和板。 <ol><li>准备在15毫升管9毫升的DMEM +中。 </li><li>从液氮罐中取出一小瓶冷冻293股，并在37℃水浴小瓶，直到大多数（但不是所有）的细胞解冻。 </li><li>从第1步15毫升管取出细胞冷冻小瓶和地方。 </li><li>在180克离心5分钟，然后弃去上清液。 </li><li> 10毫升的DMEM +中重悬细胞，并转移到一个明胶包100毫米盘。在37℃，5％的CO 2培养箱中孵育，直到他们80-90％融合的细胞。 </li><li> 293细胞中的一段话：用PBS洗细胞，吸出PBS，并添加4毫升，每10厘米0.05％trypsin/0.53 mM EDTA的菜，和孵育1分钟，在37 ° C。 </li><li>分离细胞从菜敲击，用10 ml的DMEM +中悬浮，并转移至15毫升管。在180克离心5分钟，吸出上清液。 </li><li>添加适当体积的DMEM +培养基，吹打向上和向下几次分手成单细胞悬液的细胞。 </li><li>计数的细胞数量，并调整浓度为8 × 10 5细胞每毫升与FP介质。 </li><li> 8x10的6个细胞，每100毫米培养皿（10毫升）的种子细胞，孵育37℃过夜℃，5％的CO 2。 </li></ol>第2步： 慢病毒质粒和收获病毒的转染293细胞<ol><li>对于每一个10厘米的板，使用10微克的FUW tetO基因（OCT4，SOX2，KLF4和c - Myc基因）与PMD - G 2.5ug的慢病毒的骨干和7.5微克pPax2。 </li><li>虽然aliquoting成筒3种质粒，提供Fugene 6转染试剂30μl不含血清的DMEM500μL成为第二个管和混合攻手指和孵化轻轻地在室温下5分钟。 </li><li>新增的DNA组合到Fugene 6/DMEM-containing管降落，轻轻混匀手指敲击和20分钟的孵育。 </li><li> DNA / Fugene 6复杂滴加添加到293的菜，孵育过夜，于37℃，5％的CO 2。 </li><li>第二天早上，吸出转染试剂，培养基，添加5毫升的新鲜胚胎干细胞的培养基，细胞返回到孵化器。 </li><li>在转染后48小时和72小时，收集从293中使用了10毫升的一次性无菌注射器，它通过一个0.45微米孔径的醋酸纤维素过滤器过滤，并转移到15毫升管。 </li><li>超速离心浓缩病毒上清。重悬在病毒颗粒所需的量和平等的介质，含Oct-3/4-，Sox2基因的，KLF4和c - Myc的慢病毒amixture。 </li><li>同时使病毒，准备在10厘米的菜肴Oct4-neo/rtTA或Oct4-GFP/rtTA MEFs（通过&lt;3）至90％汇合（2 × 10 6细胞每道菜） </li><li>吸用10毫升的PBS的培养液和清洗。 </li><li>倒掉PBS中，添加1毫升每碟0.05％trypsin/0.53 mM的EDTA，37 ° 10分钟。 </li><li>加入9毫升培养液，暂停一个单细胞的细胞，然后转移到一个15毫升管。 </li><li>计数的细胞数量，并调整浓度为每毫升8x10的4细胞。 10 ml的细胞悬液（8 × 10 5细胞）转移到10厘米的菜涂用明胶。菜隔夜在37℃，5％的CO 2。 </li><li>从成纤维细胞菜吸培养基，添加10毫升的含有病毒的媒介。孵育4小时通宵细胞在37℃，5％的CO 2。 </li><li> 24后吸出从成纤维细胞菜的培养基，添加10毫升的新鲜胚胎干细胞的培养基。 </li><li>定期ES细胞培养基更换培养基中含有强力霉素启动的四个基因的表达，换句话说，启动重新编程。 </li><li>如果使用一个Oct4的新记者，然后启动随时药物的第6和第9天之间。 </li><li>更改媒体每天的殖民地，直到变得足够大，被拾起。殖民地首先应该成为可见约一周后开始重整。他们应该变得足够大，要20天左右采摘。 </li></ol>第3步： 撑起的iPS殖民地采摘前的一天： <ol><li>种子明胶包被的24孔板所需数量与γ辐照DR4的MEFs </li></ol>选择在第一天的克隆： <ol><li>饲料板块的殖民地，采摘前1-2小时。 </li><li>加入15μl的PBS的一个V型底的96孔盘的水井。 </li><li>含有殖民地，一旦被拾起，用PBS洗净的菜，菜中添加的PBS10毫升。 </li><li>挑选小5μL吸管的技巧（4μLP20 pipetman）殖民地。转让合作在96盘lonies良好。 </li><li>加入20μL胰蛋白酶每口井使用的多通道（12）pipetor。吸取最多下降了3倍。 37 ° 4分钟。 </li><li> 100μL的ES培养基中添加胰酶消化菌落。吸取最多下降了10倍。从96菜6克隆一次传输24孔盘，使用在其他位置上每12个地方pipetor的提示。 </li><li> 24孔板中的细胞，待细胞达到80％-90％汇合，每日喂养与胚胎干细胞的培养基中生长在37℃，5％CO2培养箱。细胞可能会准备扩大在3-7天。 </li></ol>第4步<ol><li>扩大iPS细胞<ol><li>吸介质，并用1毫升的PBS细胞。 </li><li> PBS完全删除，添加0.1毫升的0.25％胰蛋白酶/ 1 mM的EDTA，并培育在37 ° C为10分钟。 </li><li>加入0.4毫升的ES介质，并暂停移液和单细胞悬液细胞。 </li><li>将细胞悬液转移到6孔板以及，加入1.5毫升的ES细胞培养基，孵育在37℃，5％CO2培养箱，直到细胞在6孔板达到80-90％汇合。在这一点上，准备冻结股票的细胞，如下所示。 </li></ol></li><li>冻结股票的制备<ol><li>吸介质，并用2毫升的PBS细胞。 </li><li> PBS完全删除，添加0.5毫升0.25％胰蛋白酶/ 1 mM的EDTA，并培育在37 ° C为10分钟。 </li><li>加入2毫升的ES介质，并暂停移液和单细胞悬液细胞。 </li><li>细胞悬液转移至15毫升管，计数的细胞数和自旋向下的细胞。 </li><li>弃上清，重悬在每毫升含2 × 10 6细胞与ES培养基中的细胞浓度。 </li><li>准备2 ES​​细胞冻存液（20％二甲基亚砜在ES培养基）和0.5％毫升小瓶分装。 </li><li> 0.5毫升细胞悬液转移冻结小瓶，轻轻混匀。 </li><li>在细胞冷冻集装箱的小瓶和保持它在-80 ° C过夜;转移到液氮长期存储的第二天。 </li></ol></li></ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Wernig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+.+In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">et al . In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epigenetic+remodeling+in+directly+reprogrammed+fibroblasts.">Global epigenetic remodeling in directly reprogrammed fibroblasts.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Brambrink, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+expression+of+pluripotency+markers+during+direct+reprogramming+of+somatic+cells.">Sequential expression of pluripotency markers during direct reprogramming of somatic cells.</a> Cell Stem Cell. 2, 151-159 (2008).</li></ol>

在这段视频中，我们演示了如何使用诱导，慢病毒系统产生GFP阳性，多能干IPS从MEFs Oct4-GFP/R26-M2rtTA和Nanog-GFP/R26-M2rtTA小鼠的细胞。此过程是一个有用的方法产生的iPS细胞，因为它允许在重组过程中的转基因表达的控制。虽然使用iPS细胞的生成病毒，阻碍了这些细胞在临床上使用，此过程确实让我们回答一些问题，强调尚未定义的重编程过程的主要生物。

<!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>Doxycycline hyclate</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>D9891-100G</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4

多能性，可以被诱导分化小鼠Oct4的病毒转导，SOX2，KLF4和c - Myc（Takahashi和Yamanaka，2006年; Wernig等人，2007年，大来，等，2007;。Maherali，等， 2007年）。我们已制定了一个重新编程的战略，这四个转录因子的表达强力霉素（DOX）诱导慢病毒载体（Brambrink等，2008）。使用这些诱导结构，我们可以推导出诱导多能干细胞（iPS）细胞从小鼠胚胎成纤维细胞（MEFs）。在这段视频中，我们展示了诱导慢病毒表达四个转录因子，并展示如何用这些病毒感染MEFs中，以产生iPS细胞的生成过程。通过使用诱导慢病毒，四个doxycyline此外培养基控制因素的表达。该系统比传统的逆转录病毒感染的优势是能够打开和关闭基因，使重新编程和基因表达的要求的动力学进行详细分析。

Watch this Scientific Journal Video about Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 at JoVE.com

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

该视频显示程序生成诱导多能干细胞诱导慢病毒表达OCT4，SOX2，c - Myc和KLF4。

从MEFS iPS细胞生成通过强制SOX - 2的表达，10月4，c - Myc基因和Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

generating-ips-cells-from-mefs-through-forced-expression-sox-2-oct-4

Research

JoVE Journal

Biology

36.2K 次观看.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. 该视频显示程序生成诱导多能干细胞诱导慢病毒表达OCT4，SOX2，c - Myc和KLF4。

视频: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

从MEFs基质胶我的MEFs传代的胚胎干细胞

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

科研

生物学

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

从MEFs基质胶2：到基质胶从MEFs分裂的胚胎干细胞

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

从MEFs基质胶3：传代的胚胎干细胞从基质胶到基质胶

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

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 ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Cryopreserving和恢复人类iPS细胞血清替代馈线中使用完整的基因敲除

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

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 ...