这项工作是支持（部分）以色列科学基金会（批准11分之399），德意志研究联合会（DFG）的框架内，德国和以色列的项目合作（DIP）和拉巴波特家庭研究所的研究工作的医学科学。

1。产生的诱导性细胞系<ol><li>实验之前，人们需要找到所有用于制备所需的细胞系的细胞死亡的药物G418的最小浓度。为此目的，板中给出的细胞在培养基中，将用于在实验过程中，添加的G418的浓度增加（0-1,000微克每毫升）在70％汇合的几个重复板。监控每天来确定的最低G418浓度，有效地杀死细胞的细胞。细胞死亡是内观察3-7天。 </li><li>在此之前产生的它建议以验证表达的shRNA的质粒瞬时转染实验，可以在一定程度上减少所研究的基因的表达的细胞株。这可以在任何可有效地转染的细胞系，完成。 </li><li>板T-REX-293细胞在每10cm的板的密度为约5×10 6个细胞在8毫升的DMEM培养基（含有10％四环素学年茎核准FBS，2mM L-谷氨酰胺，100单位每ml青霉素和0.1毫克每毫升链霉素，5微克每毫升含杀稻瘟素）。孵育过夜的板在37℃下，5％CO 2。 </li><li>翌日，改变介质8毫升新鲜培养基转染前。 </li><li>转染的细胞，用10微克pSuperior.neo + GFP质粒（ 图1）的编码ACF1或SNF2h短发夹驱动四环素诱导型H1启动子，以及稠合到GFP和由一个构PGK启动子驱动的新霉素抗性基因。添加的DNA到500μl的150 mM NaCl。加入另一等分的500微升的150 mM NaCl，2微升每微克DNA jetPIE试剂。的涡流添加的jetPIE的解决的DNA，拌匀。在室温下孵育15分钟。轻轻地移液管到10厘米的板含70％至80％融合细胞的DNA复合物。返回板的孵化器。 </li><li>第二天更换培养基用选择性培养基（DMEM培养基上述包含一个额外的500微克每毫升G418）。 </li><li>在接下来的两个星期监测的细胞，取代类似的新鲜培养基，每3-4天至菌落出现，可以不用显微镜可视化的选择性培养基。 </li><li>隔离的菌落之前，准备与选择性培养基的24孔板中。 </li><li>确定菌落，经眼，和在底部的板，使用的着色标记标示他们的位置。验证显微镜下观察，菌落分离。 </li><li>在无菌罩，吸板，轻轻地用温水PBS冲洗，吸出所有剩余的液体介质。将为3微升0.25％胰蛋白酶/ EDTA板的一个殖民地。移液器的胰蛋白酶反复，直到细胞分离，并将它们添加到24孔板中的井之一。在其他殖民地板重复此操作。 </li><li>成长的细胞在37℃下，5％CO 2，直到他们填补。然后分裂细胞来源于每个原始菌落分为三个井，每一个单独的12孔板中，并孵育过夜。 </li><li>翌日，更换培养基中含有1微克每毫升多西环素的培养基，从库存中双蒸水（1-5毫克每毫升）中制备的溶液在一个板。未经强力霉素在控制板更换介质。孵育细胞72小时，在37℃，5％CO 2。 </li><li>收获细胞处理和未经处理的井水，每个细胞克隆的蛋白提取物的制备和Western blot分析，以确定ACF1或SNF2h击倒的效率。使用抗体到ACF1或SNF2h以及α-微管蛋白的抗体，作为一个负荷控制。使用这些细胞在第三阱，留下未处理的，供选择的细胞克隆中，强力霉素除了导致减少至少50％，在所研究的蛋白质的水平扩张。冻结，这些细胞的等分试样，在90％FBS，10％DMSO的进一步使用。 </li></ol><p=“jove_title”的2级。诱导敲除和转染<ol><li>板细胞在选择性培养基中在10厘米的板。在每毫升1微克至一半的板添加强力霉素和孵育在37℃，5％CO 2的 48〜72小时（参见图4）。 </li><li> 48-72小时后，从各组的细胞trypsinize细胞，它们进行计数，和板每6厘米板在相同的介质中的1.5×10 6个细胞，带或不带强力霉素。细胞在37℃，5％CO 2过夜孵育。计划，以便使板从第2.1节中的每个组将提供的细胞126厘米板，包括两个重复的每个点的DAPI检测以及每个样品进行Western印迹分析（参见图4）的一个极板。 </li><li>在翌日的转染的细胞在6厘米的板与下列质粒的组合：一个空质粒1微克或相同的质粒编码的数量E4orf4，连同4μg的一个向量表达ACF1-GFP或SNF2h的-GFP呈现抗通过导入沉默突变，或相应的空载体和1μg质粒表达GFP（ 图4）3微克的shRNA的细胞克隆中。使用如上所述的jetPIE试剂（10微升每5微克DNA）以制备转染混合物。对于每个样品，准备三个板中的转染混合物。 </li><li> DNA与jetPIE试剂在室温下孵育15分钟后，轻轻移液等量的DNA复合物从每个转染组合到三个的6厘米板和孵育在37℃，5％CO 2过夜。 </li></ol> 3。 DAPI检测转染细胞中<ol><li>第二天，提取的蛋白质进行Western印迹分析，以确定从每个样品的一个板，ACF1或SNF2h已被有效地撞倒E4orf4有人在不同的样品中的平均。 </li><li>从板的DAPI用于吸取介质分析，洗的盘子轻轻地用PBS和添加1毫升4％的多聚甲醛在PBS准备，以覆盖细胞。应进行的多聚甲醛溶液的制备和与此化学品的细胞治疗中的化学遮光罩。在室温下孵育15分钟，无晃动。 </li><li>吸取的多聚甲醛，并用PBS洗三次，每次5分钟，在室温下摆动。保持在-20℃，并在-20℃下孵育至少1小时，加入80％的乙醇。该板可以乙醇在-20℃下保存数天，只要它们被密封良好，以防止乙醇蒸发和干燥。 </li><li>吸取乙醇，并用PBS洗涤细胞两次，一次用PBS-BT（含0.5％BSA和0.05％吐温20的PBS中），在室温下搅拌5分钟，每次摆动。 </li><li>为了防止非特异性抗体结合，阻止细胞在1ml PBS-BT缓冲液含有10％山羊血清的20分钟，同时在室温下摇动。然后洗净吨他PBS-BT细胞两次，每次洗5分钟。 </li><li>与第一抗体孵育的细胞（在我们的例子中一个E4orf4特定抗体）在1ml PBS-BT的同时，在室温下摇动1小时。然后在PBS-BT洗两次，一次在PBS中含有0.1％BSA，5分钟，每次洗涤。 </li><li>孵育细胞在1ml的PBS-0.1％BSA含有适当的辅助荧光标记的抗体和0.5微克每毫升最终浓度的DAPI，在室温下在黑暗中40分钟，同时摆动。然后用PBS洗5分钟，干好愿望，并保持板倒挂小时在一夜之间实现完全干燥。然后再装载的细胞的封面上滑动，使用Fluoromount-G溶液。保持板在黑暗中，直到准备好来计算凋亡细胞在4°C。 </li><li>问同事，以确定新的数字，计数凋亡细胞的各种样品中的各种板材可以做一个公正的方式。 </li><li>可视化的细胞下的upright在400X的放大倍数下（在空气中）或630X（浸没油中）的荧光显微镜。确定转染的细胞进行染色，并计算每个样本中的各种蛋白的表达简明或分散的细胞核用DAPI染色转染细胞内人口的数量。监视几个字段和计数总共至少为200转染的细胞。 </li><li>计算的百分比的核内的转染的细胞群体的细胞凋亡形态。重复实验过程中，重复2-3次，以计算出的各样品之间的变化的统计意义。 </li></ol> 4。代表性的成果有条件击倒的基因表达获得的菌落中，已成功的效率是可变的，这取决于所涉及的基因。在我们的手中，我们了12测试ACF1和18 SNF2h的。成功获得7殖民地图2 SH的样品的代表性板的选定的细胞克隆（ 图2A），以及一个单菌落（ 图2B），图3示出了具有代表性的印迹，用生长在72小时的强力霉素的存在或不存在的各种克隆从细胞中提取的蛋白质的制备。印迹染色到SNF2h和α-微管蛋白的抗体，作为一个负荷控制。两个克隆（数字4和5）表现出很强的降低在SNF2h后，多西环素诱导水平。应当指出的是，虽然在稳定的细胞系中的绿色荧光pSuperior.neo + GFP质粒（ 图1）用于生成编码一个新GFP融合蛋白的细胞系，是非常低的和其他GFP融合蛋白的表达瞬时引入到这些细胞可以很容易地检测到上述的背景绿色荧光。 的示意图进行的实验中的细胞克隆，满分测量确保的ACF1或SNF2h击倒E4orf4诱导的细胞死亡的效果示于图。 4。击倒的基因表达的多西环素治疗所需的时间可以有所不同，这取决于其水平应减少的蛋白质的稳定性。对于ACF1和SNF2h，72小时处理，在蛋白质水平上所需的高效击倒。 图5示出了表达E4orf4和GFP和DAPI染色的细胞核与稠或分散形态的外观表现为接受的细胞死亡的细胞的例子， 图6示出了一个代表性的实验证明的结果导致了，多西环素诱导ACF1击倒增加E4orf4刺激的细胞死亡（ 图6A）。增加并不会导致增加E4orf4水平（ 图6B）。此外，恢复ACF1强力霉素诱导的细胞削弱E4orf4毒性增加的水平<html>在未诱导的细胞（ 图6）中观察到的。 <img alt="图1" src="/files/ftp_upload/4442/4442fig1.jpg" fo:content-width="5.5in" fo:src="/files/ftp_upload/4442/4442fig1highres.jpg"/> 图1。地图的pSuperior.neo + GFP质粒。的地图显示四环素诱导的H1启动子驱动的shRNA表达和PGK启动子驱动的新GFP融合蛋白的表达。该地图是适用，从OligoEngine pSuperior手册。 <img alt="图2" src="/files/ftp_upload/4442/4442fig2.jpg" /> 图2。鉴定的新霉素抗性菌落。含有菌落的板图像（A）和（B）的单菌落示。通过以下方式获得的菌落在含有新霉素的选择性培养基中培养14天的细胞。 伊苏“&gt; <img alt="图3" src="/files/ftp_upload/4442/4442fig3.jpg" /> 图3。审核击倒效率在选定的菌落细胞新霉素的存在下，在选择的几个菌落接种于复孔。以及每个样品诱导的多西环素（+）和一个阱被留下未处理的（ - ）。蛋白质的提取72小时后诱导和层析，SDS-PAGE上。 Western blot法染色依次与抗体以SNF2h和α-微管蛋白的（作为装载控制），，并显示SNF2h在诱导和控制细胞水平。 <img alt="图4" src="/files/ftp_upload/4442/4442fig4.jpg" /> 图4。连续步骤的实验显示，包括多西环素治疗，以达到ACF1或SNF2h敲除，转染恢复ACF1或SNF2h表达或引入EMP 规划一个典型的击倒DAPI检测实验。性载体和介绍E4orf4或者其相应的空载体导入细胞，分析的结果。多西环素处理板以红色显示（DOX）和控制板被标记为蓝色。 <a href="http://www.jove.com/files/ftp_upload/4442/4442fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/4442/4442fig5.jpg" /> 图5。 E4orf4诱导的细胞死亡检测的DAPI检测细胞经历了图4中描述的各种治疗与E4orf4-特异性抗体和DAPI固定和染色，以可视化的转染细胞的细胞核。这种特殊的照片是从一个样品中含有E4orf4和控制绿色荧光蛋白。 （A）GFP。 （B）E4orf4。 （C）DAPI。 （D）合并后的图像。白色箭头标记GFP和E4orf4转染的细胞，包含细胞核与细胞凋亡形态。红色箭头标记与不规则形状不计入凋亡细胞的细胞核。星号标记的有丝分裂核或细胞核，只是划分。 <img alt="图6" src="/files/ftp_upload/4442/4442fig6.jpg" /> 图6。 ACF1击倒提高E4orf4诱导的细胞死亡。（A）-shRNA组四环素诱导的启动子表达ACF1从T-REX-293-源性细胞系的细胞被诱导，用强力霉素（DOX）或不及时治疗（阿霉素）。三天后，细胞转染与表达E4orf4（+ E4orf4）或空载体（E4orf4）与空载体，呈现耐引进沉默的shRNA质粒表达绿色荧光蛋白标记的ACF1突变。转染后，用抗体染色E4orf424小时用DAPI固定细胞。诱导的细胞死亡，测定由上述的DAPI检测和百分比òf压缩或分散核转染​​细胞进行了测定。示出了一个代表性实验重复3次，及误差棒表示标准偏差。 （B）平行板细胞收获Western blot分析。染色的抗体E4orf4，GFP和ACF1的印迹。内源性ACF1和ACF1-GFP在两个不同的面板。

<ol>
	<li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science (New York, N.Y). 296, 550-553 (2002).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+suppression+of+tumorigenicity+by+virus-mediated+RNA+interference.">Stable suppression of tumorigenicity by virus-mediated RNA interference.</a> Cancer. 2, 243-247 (2002).</li><li>Czauderna, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+shRNA+expression+for+application+in+a+prostate+cancer+mouse+model.">Inducible shRNA expression for application in a prostate cancer mouse model.</a> Nucleic acids research. 31, e127 (2003).</li><li>Matsukura, S., Jones, P. A., Takai, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+conditional+vectors+for+hairpin+siRNA+knockdowns.">Establishment of conditional vectors for hairpin siRNA knockdowns.</a> Nucleic acids research. 31, e77 (2003).</li><li>Wiznerowicz, M., Trono, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+suppression+of+cellular+genes:+lentivirus+vector-mediated+drug-inducible+RNA+interference.">Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference.</a> Journal of. 77, 8957-8961 (2003).</li><li>Brestovitsky, A., Sharf, R., Mittelman, K., Kleinberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adenovirus+E4orf4+protein+targets+PP2A+to+the+ACF+chromatin-remodeling+factor+and+induces+cell+death+through+regulation+of+SNF2h-containing+complexes.">The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes.</a> Nucleic acids research. 39, 6414-6427 (2011).</li><li>Livne, A., Shtrichman, R., Kleinberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+activation+by+adenovirus+E4orf4+protein+is+cell+line-specific+and+is+mediated+by+the+death+receptor+pathway.">Caspase activation by adenovirus E4orf4 protein is cell line-specific and is mediated by the death receptor pathway.</a> J. Virol. 75, 789-798 (2001).</li><li>Shtrichman, R., Kleinberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+type+5+E4+open+reading+frame+4+protein+induces+apoptosis+in+transformed+cells.">Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells.</a> J. Virol. 72, 2975-2982 (1998).</li><li>Shtrichman, R., Sharf, R., Kleinberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+E4orf4+protein+interacts+with+both+Ba+and+B'+subunits+of+protein+phosphatase+2A,+but+E4orf4-induced+apoptosis+is+mediated+only+by+the+interaction+with+Ba.">Adenovirus E4orf4 protein interacts with both Ba and B' subunits of protein phosphatase 2A, but E4orf4-induced apoptosis is mediated only by the interaction with Ba.</a> Oncogene. 19, 3757-3765 (2000).</li><li>Galluzzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+cell+death+in+higher+eukaryotes.">Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.</a> Cell death and differentiation. 16, 1093-1107 (2009).</li><li>Lavoie, J. N., Nguyen, M., Marcellus, R. C., Branton, P. E., Shore, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E4orf4,+a+novel+adenovirus+death+factor+that+induces+p53-independent+apoptosis+by+a+pathway+that+is+not+inhibited+by+zVAD-fmk.">E4orf4, a novel adenovirus death factor that induces p53-independent apoptosis by a pathway that is not inhibited by zVAD-fmk.</a> J. Cell Biol. 140, 637-645 (1998).</li><li>Li, 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+adenovirus+E4orf4+protein+induces+growth+arrest+and+mitotic+catastrophe+in+H1299+human+lung+carcinoma+cells.">The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells.</a> Oncogene. 28, 390-400 (2009).</li><li>Lavoie, J. N., Champagne, C., Gingras, M. -. C., Robert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+E4+open+reading+frame+4-induced+apoptosis+involves+dysregulation+of+Src+family+kinases.">Adenovirus E4 open reading frame 4-induced apoptosis involves dysregulation of Src family kinases.</a> J. Cell Biol. 150, 1037-1055 (2000).</li></ol>

没有利益冲突的声明。

敲除特定基因的表达，是一个重要的贡献的一种蛋白质调控途径的调查方法。由于构击倒必需基因的表达产生负面影响细胞的增殖，他们的研究可能需要稳定的条件击倒系统的siRNA或应用程序无论是短暂的介绍。生成的稳定的细胞系的能力对于药物引起的这里描述的shRNA表达，提供了一个优势，在许多细胞系中，这可能不是高效的瞬时转染，并在使用的病毒，需要重复制备，并有时一个额外的短长...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>高糖DMEM </td><td> Gibco公司</td><td> 41965 </td><td></td></tr><tr><td>春节体系认证FBS </td><td> Clontech公司</td><td> 631106 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td><td></td></tr><tr><td>笔链球菌</td><td> Gibco公司</td><td> 15140 </td><td></td></tr><tr><td> 0.25％胰蛋白酶-EDTA </td><td> Gibco公司</td><td> 25200 </td><td></td></tr><tr><td> T-REX-293细胞</td><td> Invitrogen公司</td><td> R710-07 </td><td></td></tr><tr><td> G418 </td><td>西格玛</td><td> A1720 </td><td></td></tr><tr><td>杀稻瘟</td><td> Invit罗根</td><td> R210-01 </td><td></td></tr><tr><td> pSuperior.neo + GFP质粒</td><td> OligoEngine </td><td> VEC-PBS-0007/0008 </td><td></td></tr><tr><td> jetPIE </td><td> Polyplus转染</td><td> 101-40 </td><td></td></tr><tr><td>多西环素</td><td>西格玛</td><td> D9891 </td><td></td></tr><tr><td>多聚甲醛</td><td>电子显微镜科学</td><td> 15710 </td><td></td></tr><tr><td> 4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI） </td><td>西格玛</td><td> D9542 </td><td></td></tr><tr><td> Fluoromount-G </td><td> SouthernBiotech </td><td> 0100-01 </td><td></td></tr></tbody></table>

preparation of cell-lines for conditional knockdown of gene expression and measurement of the knockdown effects on e4orf4-induced cell death

的研究是非常重要的贡献感兴趣的各种途径的1,2的蛋白质在哺乳动物细胞中的基因表达的功能失活。然而，有条件击倒的基因的表达是必需的情况下，当没有被细胞的耐受性，构击倒了很长一段的时间3-5。在这里，我们描述了一个协议，准备允许有条件的ACF染色质重塑因子亚基敲除的细胞株。这些细胞株诱导的细胞死亡的ACF的贡献便于确定由腺病毒E4orf4蛋白6。编码为ACF1和SNF2h亚基的ACF染色质重塑因子的短发夹RNA的序列进行克隆到一个多西环素诱导型启动子中还含有新霉素抗性基因的基因的质粒的旁边。选择的新霉素抗性细胞克隆在G418的存在下，和隔离。将所得的细胞系诱导的强力霉素治疗，而一旦​​ACF1或SNF2h表达水平降低，细胞转染的质粒编码E4orf4或空载体。确认具体的shRNA结构的影响，ACF1或SNF2h蛋白水平恢复到WT水平的转染与表达质粒ACF1或SNF2h呈现抵抗的shRNA引入沉默突变。通过DAPI染色的测定，其中的频率测定细胞核与细胞凋亡在转染的细胞群体的形貌的外观7-9 E4orf4的能力，诱导细胞死亡的各种样品中测定。 这里所描述的协议，可用于测定各种蛋白质的功能贡献的情况下诱导细胞死亡的蛋白质的合作伙伴，构击倒时，可能是电池致命的。

Watch this Scientific Journal Video about Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death at JoVE.com

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

ACF染色质重塑因子E4orf4诱导的细胞死亡的贡献进行了测量。该协议包括在多西环素治疗诱导有条件击倒的ACF的亚基ACF1和SNF2h的细胞克隆的选择，使用的DAPI检测测量E4orf4-诱导的细胞死亡的诱导的细胞系。

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

preparation-cell-lines-for-conditional-knockdown-gene-expression

Research

JoVE Journal

Biology

10.9K 次观看.  Technion - Israel Institute of Technology. ACF染色质重塑因子E4orf4诱导的细胞死亡的贡献进行了测量。该协议包括在多西环素治疗诱导有条件击倒的ACF的亚基ACF1和SNF2h的细胞克隆的选择，使用的DAPI检测测量E4orf4-诱导的细胞死亡的诱导的细胞系。

视频: Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

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 ...

科研

生物学

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...