这项工作是由武田科学基金会（TF）的支持;旭硝子基金会;上原纪念基金会（HF）;在仓田纪念日立科学技术基金会（TF和HF）;格兰特提供的援助青年科学家（B）（＃25830131），格兰特 - 在急救科学研究（C）（＃15K06895）（TF）;和格兰特 - 在急救科学研究的创新领域"转录周期"（＃25118512＆＃15H01354），格兰特 - 在急救科学研究（B）（＃15H04329），格兰特提供的援助的探索性研究（ ＃26650059）和"基因组支持"（＃221S0002）（HF）由教育部，文化，体育，科学和技术的日本。

工程DNA结合分子认识到目标轨迹的1.设计<ol><li>对于使用CRISPR复合enChIP，使用CRISPRdirect网络工具（http://crispr.dbcls.jp），以确定所关注形容为先前18的基因组区域的候选gRNA靶序列。此Web工具将返回表格23 bp的基因组位点5&#39;-N 20 NGG-3&#39;的目标区域内。 </li><li>合成一个gBlock包括U6启动子序列和5&#39;- N 20中5&#39;-N 20 NGG-3&#39;使用商业服务的序列（ 见图 2）19,20。用于亚克隆的目的，包括gBlock以外适当的限制性酶位点。 </li><li>插入gBlock到适当的载体如先前所述16。对于gBlocks几个逆转录病毒载体（请参阅材料 ）。 </li><li>对于使用TAL蛋白enChIP，设计TAL蛋白识别TARG等位点。生成质粒编码使用商业服务TAL蛋白。产生含有稠合有一个或多个标记，如3×FLAG如前所述15的TAL蛋白质的表达载体。 </li></ol> 2.建立细胞的enChIP分析<ol><li>表达3×FLAG-dCas9和gRNA（CRISPR基于程序）或3×FLAG-TAL（TAL蛋白质基过程）在细胞中被如先前所描述的14-17进行分析。 <ol><li>使用瞬时转染，如果转染效率是高的。可含有3×FLAG-dCas9和CMV启动子的表达载体（ 见材料）。 </li><li>考虑建立稳定的转化使用常规方法，如果瞬时转染效率是低的。除了 ​​为gBlock上述逆转录病毒载体，是3×FLAG-dCas9的反转录病毒载体可用（ 参见材料）。 </li></ol></li><li&gt;通过使用如先前14-17描述的抗FLAG抗体（抗体）免疫印迹分析确认3XFLAG-dCas9或3XFLAG-TAL蛋白在转染细胞中的表达。 注意:这些标记的蛋白质的表达，也可以通过细​​胞内染色用抗FLAG-FITC和随后的FACS分析证实。一种协议，用于细胞内染色可以从我们的主页（http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/iChIP_protocols/english.html）下载。 </li><li>确认gRNA通过标准的RT-PCR法表达。 </li><li>蛋白质与感兴趣的基因组区域相互作用的定量识别，可以考虑使用稳定同位素标记的通过在细胞培养物（SILAC）分析结合enChIP（enChIP-SILAC）氨基酸。 注:媒体的SILAC分析，可以从商业供应商处购买。 SILAC是在检测特异性相互作用强大。 <ol><li>培养细胞SILAC重介质。准备细胞文化前进红色在SILAC光介质作为阴性对照。使用至少5×10 7个细胞的每个SILAC介质（重或轻）。为有效的标记，同时添加L-赖氨酸2HCl的，13 C 6和L-精氨酸盐酸，在SILAC重媒体13 C 6，15 N 4。注:至少有五细胞分裂所必需的标签蛋白。 <ol><li>例如，培养人纤维肉瘤HT1080细胞稳定表达3XFLAG-dCas9和gRNA在37℃和5％的CO 2在DMEM培养基SILAC和透析的胎牛血清与赖氨酸2HCl的加L-精氨酸盐酸（光介质）或13 C 6 L-赖氨酸二盐酸盐加13 C 6 15 N 4 L-精氨酸盐酸（重介质）（ 见材料）16。添加50毫克轻或重的L-赖氨酸-2HCl的和L-精氨酸盐酸在500毫升培养基中。 </li><li> Trypsinize和replate细胞才汇合，使细胞保持指数growtH。 </li></ol></li></ol></li></ol> 3.交联细胞与甲醛<ol><li>对细胞悬浮培养，转移2×10 7细胞表达3XFLAG-TAL蛋白质或3XFLAG-dCas9加gRNA和暂停在30ml常规培养基在50ml离心管中。当使用更多的细胞，提高试剂的数量和金额比例。对于SILAC实验，混合在重介质或光介质（各5×10 7个细胞）培养的细胞和1×10 8个细胞的总分为5试管含有2×10 7细胞。 </li><li>添加810微升37％的甲醛的30毫升细胞悬浮液（最终浓度1％），并在37℃下5分钟。对于贴壁细胞，直接通过加入810微升37％的甲醛至30ml培养基中固定的细胞在培养皿中。 </li><li>停止交联通过加入3.1毫升1.25M的甘氨酸溶液（最终浓度127毫摩尔），和在室温下孵育10分钟。 </li><li>通过离心（300×g下5分钟，4℃）收集细胞。小心弃去上清液，包括甲醛，并将其存储在适当的废液瓶。 <ol><li>对于贴壁细胞，分离细胞用细胞刮刀和收获在50ml管中，并作为本步骤中所述收集细胞。 </li><li>对于SILAC实验，混合在重介质或光介质（5×10 7个细胞每）培养分离的细胞，1×10 8个细胞的总分为5试管含有2×10 7细胞，并如在本描述的收集细胞步骤。 </li></ol></li><li>每管洗细胞沉淀用30ml的PBS两次。小心弃去上清液，包括甲醛，并根据化学品的安全指导方针将其存储在bottle.Handle甲醛废适当的废物。固定的细胞可以冷冻并储存于-80℃。 </li></ol> 4.制备染色质（每2×107细胞 ） <ol><li>暂停固定的细胞在10毫升细胞裂解缓冲液（10mM的Tris（pH 8.0）中，1毫摩尔EDTA，0.5％IGEPAL CA-630，和1×蛋白酶抑制剂），并在冰上孵育10分钟。 </li><li>离心样品（830×g下在4℃，8分钟），小心弃去上清液。 </li><li>暂停沉淀在10ml核裂解缓冲液（10毫摩尔Tris（pH值8.0），1mM EDTA中，0.5M氯化钠，1％的Triton X-100，0.5％脱氧胆酸钠，0.5％月桂酰肌氨酸，和1x蛋白酶抑制剂）。冰上孵育10分钟，并涡旋振荡每2-3分钟。 </li><li>离心样品（830× 在4℃，8分钟g），并小心地弃去上清液。 </li><li>在10ml PBS中洗涤沉淀。将沉淀（染色质级分）可以储存在-80℃之后在液态氮立即冻结。 </li></ol> 5.超声处理的染色质（每2×10 7细胞） <ol><li>暂停在800微升的染色质部分改进的裂解缓冲液3（10毫摩尔Tris（pH值8.0），1mM EDTA中，150 mM氯化钠，0.1％脱氧胆酸钠，0.1％SDS中，和1x蛋白酶抑制剂），并转移到1.5ml试管。 </li><li>超声处理使用超声波仪（参见材料）的染色质和条件如下:输出:3;税:100％（连续）;时间:自由。执行超声处理10-18个循环持续10秒和在冰上冷却20秒。为了避免过热，孵育样品在冰上2分钟，每5-6个周期。为了避免起泡，保持声处理探针0.5厘米管子的底部上方的尖端的位置。 </li><li>离心样品（16,000×g下在4℃下进行10分钟），将上清转移到1.5ml试管。将超声处理的染色质可以储存在-80℃之后在液态氮立即冻结。 </li></ol> 6.评估染色质碎片的<ol><li>混合10微升片段化的染色质与85微升蒸馏水。 </li><li>加入4微升和的5 M氯化钠孵育在65℃CO / N。 </li><li>加入1微升10毫克/毫升RNA酶A和孵育在37℃下45分钟。 </li><li>加入2微升的0.5M EDTA（pH 8.0）中，4微升的1M Tris（pH6.8）中，和1微升20mg / ml的蛋白酶K，再孵育在45℃下1.5小时。 </li><li>单独的10微升样品通过电泳中不包含染色染料，如SYBR绿1％琼脂糖凝胶。 </li><li>染色凝胶来评价零散染色质的长度的分布。产生0.5-2 kbp的（范围0.2-4千碱基对）的平均长度条件，推荐。 </li><li>纯化通过苯酚-氯仿提取或通过使用DNA提取试剂盒（ 参见材料）从其余样品的DNA。将纯化的DNA可以作为输入的DNA来估计enChIP分析的产率（见8.11）。 </li></ol> 7.制备磁珠共轭与抗体（每2×10 7细胞） <ol><li>预削2 1.5 ml的管，一个用于使用正常小鼠IgG预澄清，另一个用于孵育抗FLAG抗体。添加150微升蛋白G偶联的磁珠（参见材料），以每管。 </li><li>将管上的磁体支架，等待3分钟。弃去上清液吹打。 </li><li>暂停在1ml PBS中含有0.01％Tween-20（PBS-T）的珠。将管上的磁性底座，等待2分钟。弃去上清液吹打，然后重复步骤。 </li><li>暂停在1ml含有0.1％BSA的PBS-T中的珠子。 </li><li>加入15微克正常小鼠IgG或抗FLAG抗体和旋转4°CO / N。 </li><li>后短暂离心，然后将管上的磁体支架，等待3分钟。弃去上清液吹打。 </li><li>暂停在1ml的PBS-T中的珠子。反转样品几次后短暂离心。将管上的磁体支架，等待3分钟，然后通过移液丢弃上清液。重复洗两次用PBS-T（总共三个洗涤步骤）。 </li></ol> 8.染色质免疫沉淀（每2×10 7细胞） <ol><li>混合在5.3中制备的分散的染色质）（约800微升）与五分之一体积的（大约200微升含5％的Triton X-100（1％的Triton X-100的最终浓度）改进的裂解缓冲液3）。 </li><li>添加染色质溶液，以在其中结合有正常小鼠IgG蛋白G偶联的磁珠制备的管。旋转，在4℃下进行1小时。 </li><li>将管上的磁体支架，等待3分钟。 </li><li>将上清液转移到在其中结合有抗FLAG抗体蛋白G偶联的磁珠制备的管。旋转，在4°CO / N。 </li><li>将管上的磁体支架，等待3分钟。弃去上清液吹打。 </li><li>暂停在1ml的低盐缓冲液（20mM的Tris（pH 8.0）中，2毫摩尔EDTA，150毫摩尔NaCl，1％三珠吨X-100，0.1％SDS中，和1x蛋白酶抑制剂），并旋转，在4℃下5分钟。将管上的磁体支架，等待3分钟。弃上清，吹打，重复清洗步骤。 </li><li>洗用高盐缓冲液（20mM的Tris（pH 8.0）中，2毫摩尔EDTA，500毫摩尔NaCl，1％的Triton X-100，0.1％SDS中，和1x蛋白酶抑制剂）的两倍珠。 </li><li>洗用的LiCl缓冲液（10mM的Tris（pH 8.0）中，1毫摩尔EDTA，250毫的LiCl，0.5％IGEPAL CA-630，0.5％脱氧胆酸钠，和1x蛋白酶抑制剂）的两倍珠。 </li><li>洗用TBS-IGEPAL CA-630（50毫摩尔Tris（pH值7.5），150 mM氯化钠，0.1％IGEPAL CA-630，和1x蛋白酶抑制剂）一旦珠。 </li><li>暂停在200μl洗脱缓冲液的珠（50毫摩尔Tris（pH值7.5），150 mM氯化钠，0.1％IGEPAL CA-630，1×蛋白酶抑制剂，和500微克/毫升3×FLAG肽）和在37℃下20分钟。将管上的磁体支架，等待3分钟。将上清液转移到一个新的1.5毫升管，并重复ELUT离子一步。 </li><li>净化洗脱液通过苯酚-氯仿提取或通过使用DNA提取试剂盒的DNA小部分（例如，5％）（见材料）。将纯化的DNA可用于PCR用特定引物集来估计产量enChIP分析由与如先前14,16所述制备步骤6.7输入的DNA进行比较。 </li></ol> 9. SDS-PAGE，染色，质谱分析<ol><li>混合洗脱物（400微升）用1毫升2-丙醇，加入50μl3M乙酸钠（pH 5.2）和5微升20毫克/毫升糖原。沉淀的染色质在-20℃CO / N。 </li><li>离心样品（16,000×g下在4℃下30分钟）并弃去上清液。冲洗用1ml 70％乙醇，离心再次的粒料（16,000×g下在4℃下进行10分钟）。吹打完全弃上清。 </li><li>暂停在40微升2×样品缓冲液（125毫摩尔Tris（pH6.8）中，10％的2-MERC沉淀aptoethanol，4％SDS，10％蔗糖和0.004％溴酚蓝）。涡旋5分钟，以完全溶解沉淀，然后孵育在100℃下30分钟以变性蛋白和逆转交联。 </li><li>对于SDS-PAGE凝胶上的运行40微升样品直至染料达到1厘米以下的井。注意:我们通常使用4-20％梯度凝胶，但其他％凝胶可以使用。 </li><li>染色用考马斯亮蓝或银染的凝胶。 </li><li>切胶成五片（2 mm高）。 </li><li>如先前13-16说明进行凝胶消化和质谱分析。 </li><li>对于SILAC实验用5×10 7个细胞每（总共1×10 8个细胞），从最初的5管在40微升2×样品缓冲液（这是没有必要按比例增加）暂停粒料。 </li></ol> 10.纯化RNA和RNA测序分析<ol><li>为了净化enChIP后的RNA，加酶抑制剂的5单位/毫升（SEë材料），以所有的缓冲溶液，除改进的裂解缓冲液3和洗脱缓冲液中，向其中添加RNA酶抑制剂的40单位/毫升。 </li><li>混合洗脱物（400微升），16微升的5M NaCl和孵育在65℃下2小时。 </li><li>加入1毫升酸 - 胍 - 酚系试剂（参见材料）到样品中。涡旋15秒，然后在室温下孵育5-15分钟。离心样品15分钟，12000×g下和室温。 </li><li>上清转移到一个新的1.5毫升管，加入5微升对溴苯甲醚的。涡旋15秒，然后在室温下孵育3-5分钟。离心样品以12,000×克和RT 10分钟。 </li><li>转移上清液（约1毫升）到新的2ml管中，加入1毫升2-丙醇。倒置该管并在室温下孵育10分钟。装载到混合物中的RNA纯化试剂盒的柱（ 参见材料）。离心列在12000×1分钟 G和RT。 </li><li>洗用400μl的RNA洗涤缓冲液中的RNA纯化试剂盒（参见材料）的列。 </li><li>的5微升DNA酶I（1U /微升），8微升10×DNA酶I反应缓冲液，3微升DNA酶/无RNase水，和64微升的RNA洗涤缓冲液中添加80μl的DNA酶I的鸡尾酒（混合物的RNA纯化试剂盒（参见材料））到柱上。孵育样品在37℃下进行15分钟，然后在12000×g下和RT离心30秒。 </li><li>用400微升RNA预洗缓冲液中的RNA纯化试剂盒（见材料）的两倍洗列。 </li><li>洗脱用50μlDNA酶/无RNase水的核糖核酸。将洗脱的RNA可用于RNA测序。 </li></ol>

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Biotechnol. 3, 626-629 (2012).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locus-specific+biochemical+epigenetics+/+chromatin+biochemistry+by+insertional+chromatin+immunoprecipitation.">Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immunoprecipitation.</a> ISRN Biochem. 2013, 913273 (2013).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+retaining+molecular+interactions+by+the+iChIP+system+using+recombinant+exogenous+DNA-binding+proteins.">Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins.</a> BMC Mol. Biol. 15, 26 (2014).</li><li>Fujita, T., Kitaura, F., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+of+the+Thy28-MYH9+axis+in+B+cell-specific+expression+of+the+Pax5+gene+in+chicken+B+cells.">A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells.</a> PLoS One. 10, (2015).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+proteins+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Biochem. Biophys. Res. Commun. 439, 132-136 (2013).</li><li>Fujita, 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=Identification+of+telomere-associated+molecules+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP).">Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).</a> Sci. Rep. 3, 3171 (2013).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+associated+with+an+IFN&#947;-responsive+promoter+by+a+retroviral+expression+system+for+enChIP+using+CRISPR.">Identification of proteins associated with an IFN&#947;-responsive promoter by a retroviral expression system for enChIP using CRISPR.</a> PLoS One. 9, e103084 (2014).</li><li>Fujita, T., Yuno, M., Okuzaki, D., Ohki, R., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+non-coding+RNAs+associated+with+telomeres+using+a+combination+of+enChIP+and+RNA+sequencing.">Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing.</a> PLoS One. 10, e0123387 (2015).</li><li>Naito, Y., Hino, K., Bono, H., Ui-Tei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPRdirect:+software+for+designing+CRISPR/Cas+guide+RNA+with+reduced+off-target+sites.">CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites.</a> Bioinformatics. , (2014).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Esvelt, K. 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=Orthogonal+Cas9+proteins+for+RNA-guided+gene+regulation+and+editing.">Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.</a> Nat. Methods. 10, 1116-1121 (2013).</li><li>Qi, L. S., 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=Repurposing+CRISPR+as+an+RNA-guided+platform+for+sequence-specific+control+of+gene+expression.">Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.</a> Cell. 152, 1173-1183 (2013).</li><li>Wu, X., 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=Genome-wide+binding+of+the+CRISPR+endonuclease+Cas9+in+mammalian+cells.">Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.</a> Nat. Biotechnol. 32, 670-676 (2014).</li><li>Kuscu, C., Arslan, S., Singh, R., Thorpe, J., Adli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+characteristics+of+off-target+sites+bound+by+the+Cas9+endonuclease.">Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.</a> Nat Biotechnol. 32, 677-683 (2014).</li><li>Cencic, 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=Protospacer+adjacent+motif+(PAM)-distal+sequences+engage+CRISPR+Cas9+DNA+target+cleavage.">Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.</a> PLoS One. 9, 109213 (2014).</li><li>O'Green, H., Henry, I. M., Bhakta, M. S., Meckler, J. F., Segal, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+analysis+of+Cas9+binding+specificity+using+ChIP-seq+and+targeted+sequence+capture.">A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.</a> Nucleic Acids Res. 43, 3389-3404 (2015).</li><li>de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shelterin:+the+protein+complex+that+shapes+and+safeguards+human+telomeres.">Shelterin: the protein complex that shapes and safeguards human telomeres.</a> Genes Dev. 19, 2100-2110 (2005).</li></ol>

The authors have filed a patent on enChIP (Patent name: 'Method for isolating specific genomic regions using DNA-binding molecules recognizing endogenous DNA sequences'; Patent number: PCT/JP2013/74107). H.F. is a member of the Advisory Board of Addgene.

Here, we describe the purification of specific genomic regions using engineered DNA-binding molecules such as the CRISPR system and TAL proteins, and the identification of proteins and RNAs bound to these genomic regions. Binding of engineered DNA-binding molecules to the genome may affect chromatin structure, including nucleosome positioning, and may abrogate genomic functions, as described in CRISPR interference experiments21. To avoid these potential aberrant effects, we propose specific guidelines for choosing target genomic regions. First, to avoid potential inhibition of the recruitment of RNA polymerases and transcription factors, as well as disruption of nucleosome positioning around the transcription start site, the target regions for analyses of promoter regions should be several hundred base pairs upstream of (5' to) the transcription start site. By contrast, when analyzing genomic regions with distinct boundaries, such as enhancers and silencers, genomic regions that are directly juxtaposed to these regions can be targeted because it is less likely that the binding of engineered DNA-binding molecules will affect their functions. Furthermore, it is best to avoid using target regions that are conserved among different species, because important DNA-binding molecules often bind to evolutionarily conserved regions and inhibition of their binding might disrupt the functions of the target genomic regions. In this regard, it is always necessary to check that the function of the target genomic region is maintained in the established cells used for enChIP analyses. Because multiple gRNAs can be tested easily and it is tedious and expensive to generate multiple versions of TAL or zinc-finger proteins recognizing different target genomic regions, enChIP using CRISPR is more advantageous than enChIP using other proteins.
It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites22-25. There are several ways to manage contamination of molecules bound to those off-target sites. First, the use of several, at least two, different gRNAs would be recommended. Those molecules commonly observed in enChIP using distinct gRNAs would be true positives. Second, comparison of different conditions for enChIP would be effective in cancelling contamination of non-specific molecules and molecules bound to off-target sites. Examples of those comparison sets would be (i) stimulation (-) and (+), or (ii) different cell types such as T cells vs. B cells. Finally, quantitative analysis of binding of candidate molecules should be performed to confirm their specific binding to the target sites. It is preferable to prepare cells expressing only dCas9 but not gRNA as a negative control.
Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3)14-17. However, this technique failed to detect some other known proteins interacting with these regions. For example, STAT1 reportedly associates with the IRF-1 promoter upon IFN&#947; stimulation8, but our enChIP-SILAC analysis did not detect STAT1 as a protein induced to interact with this genomic region16. In addition, in the enChIP-MS analysis of telomeres, we did not detect shelterin proteins consisting of TRF-1 and TRF-215, which have been shown to interact with telomeres26. There are a few potential reasons for these discrepancies. First, the stoichiometry of binding of Stat1 to the IRF-1 promoter might be very low. It is reasonable that enChIP-MS, including enChIP-SILAC, detects proteins that are more abundantly associated with target genomic regions; hence, the analysis of more cells might be necessary to detect these proteins. Increases in the sensitivities of MS instruments would also contribute to the efficient detection of proteins with low stoichiometric binding. Second, some proteins, possibly including Stat1 and shelterins, might be difficult targets for MS analyses. Third, in our analysis of telomere-binding proteins15, the 3&#215;FLAG-TAL proteins recognizing telomeres (3&#215;FN-Tel-TAL) might have blocked the binding of shelterins to telomeres in a competitive fashion.
In contrast to the relative difficulty of detecting transcription factors binding to specific genomic regions using enChIP, we successfully identified epigenetic regulators such as histone modification enzymes using enChIP analyses. The success of this technique may be due to the fact that epigenetic regulators bind to a broad range of genomic regions; hence, more proteins per genomic region are available for MS. Because epigenetic regulators are increasingly recognized as important targets for drugs against intractable diseases such as cancer, enChIP would be a useful tool for the identification of epigenetic drug targets.

与感兴趣的特定基因组区域相关联的分子的识别，需要理解的基因组功能，例如转录和表观遗传调控调节的机制。虽然几种技术已经开发了用于特定的基因组区域1-7中的生化分析，它们没有被广泛使用，因为其固有的问题，如有限的应用程序（例如，只对于高拷贝数或基因座基因座与重复），并在此阶段太多的时间和努力需要。 为了容易地执行特定的基因组区域的生化分析，我们已经开发了两种基因座特异性染色质免疫沉淀技术，即插入的ChIP（Internet控制器）8-13和工程化DNA结合分子介导的ChIP（enChIP）14-17 。在Internet控制器，感兴趣的基因座是由标记的外源DNA结合蛋白如莱克斯插入识别序列答轨迹然后通过使用标记的DNA结合蛋白的亲和纯化分离。在enChIP，工程化DNA结合分子，如锌指蛋白，转录激活物状（TAL）的蛋白质，和群集定期相互间隔短回文重复序列（CRISPR）配合物，用于标记的感兴趣位点（ 图1）。随后，将基因组区域被标记的DNA结合分子的亲和纯化分离。 之一的enChIP过Internet控制器的一个优点是，插入的外源DNA结合蛋白的识别序列是没有必要的。靶向使用CRISPR络合物选自Cas9（dCas9）的无催化活性的形式的和导向的RNA（gRNA）位点比这些区域通过Internet控制器或enChIP使用TAL和锌指蛋白靶向容易得多。这里，我们描述了一步一步的协议enChIP与质谱联用和RNA测序（RNA片段）识别位点Associa大泰德蛋白质和RNA的分别。

<table border="0" cellpadding="0" cellspacing="0" width="964">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:239px;">gBlock synthesis service</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">Gene Synthesis by GeneArt</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:239px;">gBlock synthesis service</td>
			<td style="width:199px;">IDT (Integrated DNA Technologies)</td>
			<td style="width:175px;">gBlocks Gene Fragments</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-neo</td>
			<td>Addgene</td>
			<td style="width:175px;">51128</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-GFP</td>
			<td>Addgene</td>
			<td style="width:175px;">51134</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-DsRed-Express2</td>
			<td>Addgene</td>
			<td style="width:175px;">51135</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-hCD2</td>
			<td>Addgene</td>
			<td style="width:175px;">51143</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">TAL synthesis service</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">GeneArt Precision TALs</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">3xFLAG-dCas9/pCMV-7.1</td>
			<td>Addgene</td>
			<td style="width:175px;">47948</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-puro</td>
			<td>Addgene</td>
			<td style="width:175px;">51240</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-IG</td>
			<td>Addgene</td>
			<td style="width:175px;">51258</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-I2</td>
			<td>Addgene</td>
			<td style="width:175px;">51259</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-neo</td>
			<td>Addgene</td>
			<td style="width:175px;">51260</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">anti-FLAG M2 Ab</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:175px;">F1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">FITC-conjugated anti-FLAG M2</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:175px;">F4049</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">DMEM medium for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89985</td>
			<td>Other medium can be purchased from Life Technologies</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">Dialyzed FBS for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89986</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">L-Lysine-2HCl for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89987</td>
			<td>for Light medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">L-Arginine-HCl for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89989</td>
			<td>for Light medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">L-Lysine-2HCl, 13C6 for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89988</td>
			<td>For Heavy medium</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:239px;">L-Arginine-HCl, 13C6, 15N4 for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89990</td>
			<td>For Heavy medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Complete, mini, EDTA-free</td>
			<td style="width:199px;">Roche Diagnostics</td>
			<td style="width:175px;">4693159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ultrasonic Disruptor UD-201</td>
			<td style="width:199px;">Tomy Seiko</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChIP DNA Clean &#38; Concentrator</td>
			<td style="width:199px;">Zymo Research</td>
			<td style="width:175px;">D5205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Dynabeads-Protein G</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">DB10004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">RNasin Plus RNase Inhibitor</td>
			<td style="width:199px;">Promega</td>
			<td style="width:175px;">N2611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Isogen II</td>
			<td style="width:199px;">Nippon Gene</td>
			<td style="width:175px;">311-07361</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Direct-zol RNA Miniprep kit</td>
			<td style="width:199px;">Zymo Research</td>
			<td style="width:175px;">R2050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">LTQ Orbitrap Velos</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">nanoLC</td>
			<td style="width:199px;">Advance, Michrom Bioresources</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">HTC-PAL autosampler</td>
			<td style="width:199px;">CTC Analytics</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
		</tr>
	</tbody>
</table>

isolation of specific genomic regions and identification of associated molecules by enchip

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

总体而言，靶基因组区域的1-30％可使用enChIP纯化。 图3包括代表数据表示enChIP的产率分析靶向端粒和干扰素（IFN）调节因子1（IRF-1）基因的启动子。作为典型的结果，表1列出实施例与在IRF-1启动子中鉴定由enChIP-SILAC， 表一的IFNγ特异性方式相关的蛋白2列出确定enChIP与质谱联用端粒结合蛋白（enChIP-MS） ，和表3列出用确定enChIP-RNA测序端粒相关联的的RNA。 <img alt="图1" src="/files/ftp_upload/53478/53478fig1.jpg" /> 图 enChIP 1.概述 。 enChIP使用CRISPR（A，B）和 TAL（C，D，E </STRONG&gt;）显示。感兴趣的轨迹被打上了设计DNA结合分子，如蛋白质TAL或由Cas9（DCAS）的催化活性形式的CRISPR系统和引导RNA（gRNA）。的分子间的相互作用是用甲醛固定或其它交联剂，如果需要的话。随后，固定的染色质通过超声处理或酶促消化分段。加标签的基因组区域是由亲和纯化来纯化。最后，交联逆转，相互作用的分子（基因组区域，RNA的，和蛋白质）均采用新一代测序和质谱鉴定。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53478/53478fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53478/53478fig2.jpg" /> 图2.序列gBlock的。U6启动子（黑色），对G核苷酸b安伏的导向序列（蓝色），引导序列（gRNA间隔物）（红色），所述支架序列（绿色），及终止子序列（橙色）被示出。 <img alt="图3" src="/files/ftp_upload/53478/53478fig3.jpg" /> 图3.产量的代表enChIP分析 。 （A）中的百分比的输入为目标的IRF-1基因座和非靶Sox2的基因座。 （B）的百分比的输入为目标端 ​​粒和非目标γ-卫星。该数字已调整，并从以前的出版物15,16修改。 <table fo:keep-together.within-page="1"><tbody><tr><td> 分类 </td><td> 蛋白质 </td></tr><tr><td>转录</td><td> DDX1，PARP1，CKAP4，Pescadillo同源，PURβ， 活化的RNA聚合酶II转录激活p15基因， BTF3，Myb的结合蛋白1A </td></tr><tr><td>组蛋白脱乙酰化， 辅阻遏物成分</td><td> RBBP4，PA2G4，TBL3 </td></tr><tr><td>乙酰转移酶</td><td>蛋白质精氨酸N-甲基转移酶1 </td></tr><tr><td> DNA拓扑异构酶</td><td> DNA拓扑异构酶2α </td></tr><tr><td>组蛋白</td><td>组蛋白H2A.Z，组蛋白H3.2 </td></tr></tbody></table> 表1:用 在确定enChIP-SILAC一个IFNγ特异性方式的人IRF-1的启动子区相关的蛋白质的例子的表已被改编从先前的出版物16。 <table fo:keep-together.within-page="1"><tbody><tr><td> 分类 </td><td> 蛋白质 </td></tr><tr><td>哺乳动物端粒结合蛋白</td><td> PML，RPA，CDK1，PARP1，PCBP1 </td></tr><tr><td>端粒碧在酵母nding蛋白质 或其他生物</td><td> IMP4 </td></tr><tr><td>与蛋白质相互作用的端粒结合 蛋白质[相关端粒结合蛋白] </td><td> DNA聚合酶α（POLA1）Cdc13p] ARMC6 [TRF2]，CTBP1 [FOXP2-POT1] exportin-5 [叔]，GNL3L [TRF1] exportin-1 [叔]，14-3-3 [叔] </td></tr><tr><td>蛋白定位于异</td><td> BEND3 </td></tr><tr><td>蛋白质调控的表观遗传标记</td><td> KDM5C </td></tr><tr><td>蛋白质的突变影响端粒功能</td><td> DNA聚合酶α（POLA1），HAT1，Nup133， CDK7，DPOE1，PRDX1，TYSY， 谷氨酸半胱氨酸连接酶，谷，SMRC1 </td></tr></tbody></table> 表2:用 确定enChIP-MS鼠标端粒相关蛋白的例子表已adapted从以前的出版物15。 <table fo:keep-together.within-page="1"><tbody><tr><td> 分类 </td><td> RNA的 </td></tr><tr><td>端粒酶组件</td><td> TERC，RMRP </td></tr><tr><td>端粒的RNA </td><td> TERRAS </td></tr><tr><td> scaRNAs </td><td> Scarna6，Scarna10，Scarna13，Scarna2 </td></tr><tr><td> H / ACA 52]形成</td><td> Snora23，Snora74a，Snora73b，Snora73a </td></tr><tr><td> C / D 52]形成</td><td> Snord17，Snord15a，Snord118 </td></tr><tr><td> lncRNA </td><td> Neat1 </td></tr></tbody></table> 表3: 具有确定enChIP-RNA的序列表已被改编自先前公布17 鼠标端粒相关的RNA的例子 。

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Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

isolation-specific-genomic-regions-identification-associated

Nanyang Technological University

Research

JoVE Journal

Biology

10.4K 次观看.  Osaka University. The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

视频: Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

的效用舞台专用中旬至下旬<em&gt;果蝇</em&gt;卵泡隔离

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...