This study was funded by Canadian Institutes for Health Research (CIHR) grant. STC holds a doctoral research award from the CIHR and the Michael Smith Foundation. EYS holds a CIHR scholarship. The CIHR Transplantation Training Program also provided graduate scholarships to STC, EYS and SS.

1.质粒DNA纯化<ol><li>使用根据制造商的协议的大量制备试剂盒提取质粒DNA。重悬的质粒DNA在500μlTE缓冲液中。 </li><li>执行苯酚：氯仿：异戊醇萃取和异丙醇沉淀以除去残留的细菌污染物。 LPS的存在干扰了转9。 <ol><li>加入500微升酚：氯仿：异戊醇（25：24：1，pH 8）中的质粒DNA，并剧烈振荡15秒。酚导致严重的皮肤灼伤和是麻醉剂使烧伤并不总是感觉，直到有严重的损害。通风柜异戊提取和使用警告：执行苯酚：氯仿。 </li></ol></li><li>为5分钟，在RT孵育混合物。离心以13,000 xg离心在室温10分钟。转移350微升上层水相的成一个新的1.5 ml收集管。 </li><li>添加350微升TE缓冲液到包含下层有机相中的管。剧烈摇晃15秒。离心以13,000×g离心5分钟，在RT。转移350微升上层水相，以同为1.5 ml收集管。 </li><li>加入70微升的3M醋酸钠（pH5.2）拌匀。加入700μl100％异丙醇。拌匀，孵育10分钟在室温。离心机以13,000×g离心10 minat 4℃。去除上清。 </li><li>加入500微升75％乙醇，以沉淀。涡旋样品，在4℃混合和离心机在5000×g离心5分钟。去除上清。的DNA是在沉淀中。 </li><li>打开管的盖和空气干燥沉淀在RT 5 - 10分钟。沉淀应成为半透明。加入500微升TE缓冲液以重悬DNA沉淀。热，在50 - 60℃下进行10分钟，以溶解DNA沉淀。 </li><li>测量260nm处的吸光度（A260）和280nm（A280）用分光计。通过用50纳克/微升乘以A260值计算DNA浓度。评估DNA的calculatin质量克A260 / A280比值。 DNA具有较高的质量应该给予1.8的A260 / A280比值 - 2.0。 </li></ol> 2.细胞培养和播<ol><li>培养RAW264.7细胞在DMEM补充有在37℃，5％CO 2培养箱9％胎牛血清（DMEM / 9％FCS）中。在每个通道，通过使用巴氏吸管连续移液分离从培养板上的细胞。通道中的细胞，每2天，和种子1.5 - 2百万个细胞在10cm组织培养物处理的培养皿作为库存。解冻的细胞每隔5的新股 - 6周。 </li><li>在转染当天，种子，每孔200,000个细胞在24孔板中的500微升DMEM / 9％FCS中的体积。孵育细胞在37℃培养箱中4小时。 </li><li>转染的细胞或者与基于脂质的试剂（步骤3.1）或蛋白质/聚胺系试剂（步骤3.2）。 </li></ol> 3.转染<ol><li>基于脂质的转染： <ol><li>热身转染reagen吨至RT在黑暗中。预热的无血清培养基和DMEM / 9％FCS中，以37℃。 </li><li>添加0.5质粒DNA微克至50μl无血清培养基的在1.5ml管中。加入2微升转染试剂与液体的表面之下的枪头。漩涡，持续6秒。 </li><li>离开在黑暗中试剂/ DNA混合物在RT下30分钟。 </li><li>在30分钟温育后，从孔中取出介质，并加入250微升新鲜的DMEM / 9％FCS中的每个孔中。 </li><li> 30分钟后，加入250微升DMEM / 9％FCS中的向试剂/ DNA混合物。通过上下吹打拌匀。 </li><li>添加300微升稀释的混合物的每个孔中。在37℃，5％CO 2培养箱4小时-孵育2。 </li><li>取出转染溶液，加入500微升DMEM / 9％FCS的。在37℃，5％CO 2的细胞刺激前孵化48小时-孵育24。 </li></ol></li><li>蛋白质/聚胺系的转染： <ol><li>热身无血清介质和DMEM / 9％FCS中，以37℃。 </li><li>添加0.75微升转染试剂以18.75微升无血清培养基中。短暂涡旋混合。在室温下孵育5分钟。加入0.5微升1微克/微升DNA导入稀释转染试剂。在室温下孵育10分钟。 </li><li>在10分钟温育后，从24孔板的每个孔中除去培养基并加入250微升新鲜的DMEM / 9％FCS的每个孔中。 </li><li> 10分钟后，加入250微升DMEM / 9％FCS中的入试剂/ DNA混合物。 </li><li>添加270微升稀释混合物到孔中。孵育在37℃，5％CO 2的培养箱中培养2 - 4小时。 </li><li>取出转染溶液，加入500微升DMEM / 9％FCS的。在37℃，5％CO 2培养箱48小时-孵育24。 </li></ol></li></ol> 4.细胞刺激和荧光素酶测定<ol><li>在很好地与250微升新鲜DMEM / 9％FCS的更换介质。 </li><li>加入50微升LPS或LPS + IL-10在所需浓度到孔中。返回板到37℃，5％CO 2培养箱。刺激2 - 6小时。 </li><li>通过抽吸除去刺激溶液，洗净用冰冷的PBS，并添加200μl的1×被动裂解缓冲液中。岩石在室温30分钟。刮和裂解物转移到1.5 ml微量离心管，并通过以20000×g离心在4℃下离心10分钟除去细胞碎片。 </li><li>转移40微升澄清的裂解液（上清液）的成白色，透明底96孔板用于根据制造商的方案的荧光素酶信号的测定。 </li><li>读取整个可见光光谱光度计荧光素酶信号。 </li></ol> 5.数据分析<ol><li>通过从各孔除以萤火虫荧光素酶的各个值由Renilla荧光素酶的值的Renilla比率：计算萤火虫。 </li><li>的处理海肾比深受：除以萤火虫计算倍数变化是的未经处理的很好。 </li></ol>

<ol>
	<li>Maurisse, R., 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=Comparative+transfection+of+DNA+into+primary+and+transformed+mammalian+cells+from+different+lineages.">Comparative transfection of DNA into primary and transformed mammalian cells from different lineages.</a> BMC Biotechnol. 10 (1), 9-18 (2010).</li><li>Thompson, C. D., Frazier-Jessen, M. R., Rawat, R., Nordan, R. P., Brown, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+for+transient+transfection+of+a+murine+macrophage+cell+line+RAW+264.7.">Evaluation of methods for transient transfection of a murine macrophage cell line RAW 264.7.</a> Biotechniques. 27 (4), 824-835 (1999).</li><li>Kim, T., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and Bioanalytical Chemistry. 397 (8), 3173-3178 (2010).</li><li>Stacey, K. J., Sweet, M. J., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+ingest+and+are+activated+by+bacterial+DNA.">Macrophages ingest and are activated by bacterial DNA.</a> Journal of immunology. 157 (5), 2116-2122 (1996).</li><li>Jiang, W., Reich, I. C., Pisetsky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+activation+of+the+RAW264.7+macrophage+cell+line+by+transfected+mammalian+DNA.">Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA.</a> Cell Immunol. 229 (1), 31-42 (2004).</li><li>Jiang, W., Pisetsky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+induction+of+HMGB1+release+from+RAW+264.7+cells+by+transfected+DNA.">The induction of HMGB1 release from RAW 264.7 cells by transfected DNA.</a> Mol Immunol. 45 (7), 2038-2046 (2008).</li><li>Hargreaves, D. C., Horng, T., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+inducible+gene+expression+by+signal-dependent+transcriptional+elongation.">Control of inducible gene expression by signal-dependent transcriptional elongation.</a> Cell. 138 (1), 129-147 (2009).</li><li>Cheung, S. T., So, E. Y., Chang, D., Ming-Lum, A., Mui, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-10+inhibits+lipopolysaccharide+induced+miR-155+precursor+stability+and+maturation.">Interleukin-10 inhibits lipopolysaccharide induced miR-155 precursor stability and maturation.</a> PLoS One. 8 (8), e71336 (2013).</li><li>Weber, M., Moller, K., Welzeck, M., Schorr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+technical+reports.+Effects+of+lipopolysaccharide+on+transfection+efficiency+in+eukaryotic+cells.">Short technical reports. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells.</a> Biotechniques. 19 (6), 930-940 (1995).</li><li>Berghaus, L. J., 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=Innate+immune+responses+of+primary+murine+macrophage-lineage+cells+and+RAW+264.7+cells+to+ligands+of+Toll-like+receptors.">Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors.</a> Comp Immunol Microbiol Infect Dis. 33 (5), 443-456 (2000).</li><li>Fiszer-Kierzkowska, A., 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=Liposome-based+DNA+carriers+may+induce+cellular+stress+response+and+change+gene+expression+pattern+in+transfected+cells.">Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells.</a> BMC Mol Biol. 12 (1), 27-36 (2011).</li><li>Jacobsen, L., Calvin, S., Lobenhofer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+effects+of+transfection:+the+potential+for+misinterpretation+of+gene+expression+data+generated+from+transiently+transfected+cells.">Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells.</a> Biotechniques. 47 (7), 617-626 (2009).</li></ol>

作者什么都没有透露。

在这里描述的协议并不仅仅着眼于转染效率，但旨在在效率的细胞的生理状态和保护之间的平衡。具体地讲，我们的过程成功地减少转染试剂的毒性和最大化的荧光素酶信号。 在协议中的一个关键步骤是在细胞的健康。杂草丛生文化不适合的转染作为其生理变化，RAW264.7细胞的一个长的时间内还可以改变单电池10的表型和功能的连续培养。刚解冻的细胞，具有低传代?...

核酸细胞转染在科研多样的应用程序。实例包括（1）的报告基因来研究不同基因元件在基因表达中的作用;（2）蛋白表达质粒过表达感兴趣的蛋白质，和（3）的小干扰RNA下调基因表达。通过操纵特定基因的表达水平和测量这种操作的微分效果，研究人员可以推导出在所选择的生物系统的基因功能。不是所有的转染方法提供相同的转染效率，甚至同一染方法不转染所有类型的细胞同等1。因此，不同的转染方法已被开发，如磷酸钙法，DEAE-葡聚糖，阳离子脂质转染，阳离子非脂质的聚合物的转染，电穿孔，核转染和2,3。 转染巨噬细胞是ESPEcially困难的，由于这一事实，即巨噬细胞是专业的吞噬细胞是给外国的材料，包括来自（甲基）的DNA 4菌非常敏感。外源DNA的引入激活Toll样受体9（TLR9）途径，导致细胞因子的产生和一氧化氮5,6。然后，这些活化巨噬细胞可能是不太适应的治疗，研究人员打算检查。 我们的实验室常规transfects与荧光素酶报告基因的RAW264.7巨噬细胞系，我们已经开发了一个协议，是足够强大的荧光素酶有比信号背景显著高，而且柔情似水的巨噬细胞，以保持其静止状态。转染的细胞的行为由萤火虫荧光素酶报告基因窝藏IκBζ（pGL3-IκBζ）的启动子区进行了评价。 IκBζ表达通过细菌细胞壁成分唇上调opolyssacharide（LPS）的7,8，和由抗炎细胞因子下调，白介素-10（IL-10）8。考虑到井之间转变体中，我们共同转染含有海肾荧光素酶基因（ 例如，phRL-TK）进行归一化目的的对照质粒。描述的协议测试各种参数，包括转染的定时，对转染试剂的类型，转染试剂和质粒DNA的量，以及转染试剂的比率，以质粒DNA后进行了优化。包括在这个协议在两个转染试剂是：（1）一个基于脂质的转染试剂和（2）一种蛋白质/聚胺系转染试剂。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PureLink HiPure Plasmid Maxiprep Kit</td>
			<td>Life Technologies</td>
			<td>K210007</td>
			<td>Any maxiprep kit will work</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:isoamyl alcohol</td>
			<td>Life Technologies</td>
			<td>15593-049</td>
			<td>Molecular Biology Grade. Phenol is toxic so work in the fume hood, if possible. Use the lower clear organic layer if two layers of liquid form in the container.</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>SH30243.01</td>
			<td>Warm in 37&#176;C water bath before use.&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Scientific</td>
			<td>SH30396.03</td>
			<td>Inactivated at 56&#176;C water bath for 45 minutes before use.</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Warm to at least room temperature before use.</td>
		</tr>
		<tr>
			<td>XtremeGene HP DBA transfection reagent</td>
			<td>Roche</td>
			<td>6366236001</td>
			<td>Warm to room temperature before use.&#160;</td>
		</tr>
		<tr>
			<td>GeneJuice</td>
			<td>EMD Millipore</td>
			<td>70967</td>
			<td>Warm to room temperature before use.&#160;</td>
		</tr>
		<tr>
			<td>5X Passive Lysis Buffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td>30 ml is included in the Dual Luciferase Reporter Assay System</td>
		</tr>
		<tr>
			<td>Dual Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfecting raw264.7 cells with a luciferase reporter gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

图1比较了在RAW264.7两个转染试剂的转染效率。所述基于脂质的试剂通常得到约25％的转染率， ​​而蛋白质/聚胺系的转染导致约5％的效率（ 图1A）。在转染效率的差异，也观察到在转染了的pGL3-IκBζ启动子报告（ 图1B）的RAW264.7细胞的荧光素酶信号。加入LPS到这些转染的细胞的增加的萤火虫萤光素酶信号，所述IκBζ启动子报告的增加转录活性的直接指示。换...

Watch this Scientific Journal Video about Transfecting RAW264.7 Cells with a Luciferase Reporter Gene at JoVE.com

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

转染RAW264.7细胞用荧光素酶报告基因

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

transfecting-raw2647-cells-with-a-luciferase-reporter-gene

Research

JoVE Journal

Biology

20.5K 次观看.  Vancouver Costal Health Research Institute. Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells. 

视频: Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

人类神经干细胞转染与Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

科研

生物学

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

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

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 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 ...

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

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

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

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

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...