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>
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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

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20.4K 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. 