Diese Arbeit wurde von Takeda Science Foundation (TF) unterstützt; die Asahi Glass Foundation; die Uehara Memorial Foundation (HF); die Kurata Memorial Hitachi Science and Technology Foundation (TF und HF); Grant-in-Aid für junge Wissenschaftler (B) (# 25830131), Grant-in-Aid for Scientific Research (C) (# 15K06895) (TF); und eine Grant-in-Aid for Scientific Research für innovative Bereiche &#39;Transcription Zyklus&#39; (# 25118512 &amp; # 15H01354), Grant-in-Aid for Scientific Research (B) (# 15H04329), Grant-in-Aid for Exploratory Research ( # 26650059) und &quot;Genome Support &#39;(# 221S0002) (HF) aus dem Ministerium für Bildung, Kultur, Sport, Wissenschaft und Technologie in Japan.

1. Design von Engineered DNA-bindende Moleküle in Anerkennung der Target-Locus <ol><li> Für enChIP Verwendung CRISPR Komplexe, verwenden Sie die CRISPRdirect Web-Tool (http://crispr.dbcls.jp) Kandidaten gRNA Zielsequenzen in der genomischen Region von Interesse, wie zuvor beschrieben 18 zu identifizieren. Dieses Web-Tool liefert 23 bp genomischer Websites der Form 5&#39;-N 20 NGG-3 &#39;im Zielbereich. </li><li> Synthetisieren eine gBlock einschließlich der U6-Promotor-Sequenz und die Sequenz der 5&#39;-N 20 in 5&#39;-N 20 NGG-3 &#39;unter Verwendung von kommerziellen Diensten (siehe 2), 19,20. Zur Subklonierung Zwecke umfassen geeignete Restriktionsenzymstellen außerhalb des gBlock. </li><li> Legen Sie die gBlock in einen geeigneten Vektor, wie zuvor beschrieben 16. Mehrere retrovirale Vektoren für gBlocks stehen zur Verfügung (siehe Materialien). </li><li> Für enChIP mit TAL-Proteinen, entwerfen TAL-Proteinen in Anerkennung der target loci. Generieren Plasmide TAL-Proteinen unter Verwendung von kommerziellen Dienstleistungen, kodiert. Generieren Expressionsvektoren, die TAL-Proteinen mit einem oder mehreren Tags wie 3 × FLAG verschmolzen, wie zuvor beschrieben 15 enthält. </li></ol> 2. Zeitpunkt der Gründung der Zellen für die Analyse enChIP <ol><li> Ausdruck 3 × FLAG-dCas9 und der gRNA (CRISPR-basierte Verfahren) oder 3 × FLAG-TAL (TAL Proteinbasis Verfahren) in den Zellen, wie zuvor beschrieben, um 14-17 analysiert. <ol><li> Verwenden transiente Transfektion, wenn die Transfektionseffizienz ist hoch. Ein Expressionsvektor, enthaltend 3 × FLAG-dCas9 und den CMV-Promotor ist verfügbar (siehe Materialien). </li><li> In Erwägung ziehen, stabile Transformanten unter Verwendung herkömmlicher Verfahren, wenn die transiente Transfektion Effizienz gering ist. Zusätzlich zu den oben genannten retroviralen Vektoren für gBlock, sind retrovirale Vektoren von 3 × FLAG-dCas9 verfügbar (siehe Materialien). </li></ol></li><li&gt; Bestätigen der Expression des 3xFLAG-dCas9 oder 3xFLAG-TAL-Proteins in den transfizierten Zellen durch Immunoblot-Analyse unter Verwendung eines Anti-FLAG-Antikörper (Ab), wie zuvor beschrieben, 14-17. Anmerkung: Die Expression dieser markierten Proteine ​​können auch durch intrazelluläre Färbung mit anti-FLAG-FITC und anschließender FACS-Analyse bestätigt werden. Ein Protokoll für die intrazelluläre Färbung steht auf unserer Homepage (http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/iChIP_protocols/english.html) heruntergeladen werden. </li><li> Ausdruck der gRNA durch Standard-RT-PCR-Technik zu bestätigen. </li><li> Für quantitative Identifizierung von Proteinen in Wechselwirkung mit der genomischen Region von Interesse, zu prüfen Verwendung eines Silac (SILAC) Analyse kombiniert mit enChIP (enChIP-SILAC). Hinweis: Medien für das SILAC Analyse kann von kommerziellen Anbietern erworben werden. SILAC ist mächtig im Nachweis von spezifischen Wechselwirkungen. <ol><li> Kulturzellen in SILAC schweren Medium. Bereiten Zellen culturot im SILAC Lichtmedium als negative Kontrolle. Verwenden Sie mindestens 5 x 10 7 Zellen pro SILAC Medium (schwer oder leicht). Für eine effiziente Markierung hinzufügen, sowohl L-Lysin-2 HCl, 13 C 6 und L-Arginin-HCl, 13 C 6, 15 N 4 in den SILAC Schwere Medien. Hinweis: Mindestens fünf Zellteilungen erforderlich, Label-Proteine ​​sind. <ol><li> Beispielsweise Kultur menschlichen Fibrosarkom-HT1080-Zellen, die 3xFLAG-dCas9 und gRNA bei 37 ° C und 5% CO 2 in DMEM-Medium für SILAC und dialysiertem fötalem Rinderserum mit Lysin-2HCl plus L-Arginin-HCl (Light-Medium) oder 13 C 6 L-Lysin-2 HCl plus 13 C 6 15 N 4 L-Arginin-HCl (Heavy-Medium) (siehe Materialien) 16. Fügen Sie 50 mg von leichten oder schweren L-Lysin-2 HCl und L-Arginin-HCl in 500 ml Medium. </li><li> Trypsinize und Ausstrich Zellen, bevor sie konfluent werden, so dass Zellen aufrechtzuerhalten exponentiellen growth. </li></ol></li></ol></li></ol> 3. Vernetzung der Zellen mit Formaldehyd <ol><li> Für Zellen in Suspensionskultur, Transfer 2 x 10 7 Zellen, 3xFLAG-TAL-Proteinen oder 3xFLAG-dCas9 zzgl gRNA und Aussetzung in 30 ml regelmäßigen Kulturmedium in einem 50 ml Zentrifugenröhrchen. Wenn mehr Zellen verwendet werden, erhöhen Sie die Mengen und Mengen von Reagenzien proportional. Für SILAC Experimenten, mischen Sie die Zellen, die in schweren Medium oder eine Lichtmedium (5 x 10 7 Zellen pro Stück) kultiviert und unterteilen die Gesamtzahl von 1 x 10 8 Zellen in 5 Röhrchen mit 2 x 10 7 Zellen. </li><li> Add 810 ul 37% Formaldehyd zu 30 ml der Zellsuspension (Endkonzentration 1%) und bei 37 ° C für 5 min. Für adhärente Zellen, direkt beheben Zellen in Kulturschalen durch Zugabe von 810 ul 37% Formaldehyd und 30 ml Kulturmedium. </li><li> Stoppen Sie die Vernetzung durch Zugabe von 3,1 ml von 1,25 M Glycin-Lösung (Endkonzentration 127 mM) undInkubation bei Raumtemperatur für 10 min. </li><li> Sammeln Zellen durch Zentrifugation (300 × g für 5 min bei 4 ° C). Vorsichtig den Überstand verwerfen einschließlich Formaldehyd und speichern sie in einem geeigneten Abfallflasche. <ol><li> Für adhärente Zellen, lösen die Zellen mit einem Zellschaber und der Ernte in einem 50-ml-Tube, und sammeln Zellen, wie in diesem Schritt beschrieben. </li><li> Für SILAC Experimenten, mischen Sie die abgelösten Zellen in schweren Medium oder eine Lichtmedium (5 x 10 7 Zellen je) kultiviert, teilen Sie die Gesamtzahl von 1 x 10 8 Zellen in 5 Röhrchen mit 2 x 10 7 Zellen, und sammeln Zellen, wie in diesem beschrieben Schritt. </li></ol></li><li> Zweimaliges Waschen des Zellpellets mit 30 ml PBS pro Röhrchen. Vorsichtig den Überstand verwerfen einschließlich Formaldehyd und speichern Sie es in einen geeigneten Abfall bottle.Handle Formaldehyd Abfall nach chemischen Sicherheitsrichtlinien. Die fixierten Zellen können eingefroren und bei -80 ° C gelagert werden. </li></ol> 4. Herstellung von Chromatin (je 2 x 107-Zellen) <ol><li> Aussetzung der fixierten Zellen in 10 ml Zelllysepuffer (10 mM Tris (pH 8,0), 1 mM EDTA, 0,5% IGEPAL CA-630 und 1 × Protease-Inhibitoren) und Inkubation auf Eis für 10 min. </li><li> Zentrifugieren Sie die Probe (830 × g bei 4 ° C für 8 min) und den Überstand verwerfen sorgfältig. </li><li> Aussetzen des Pellets in 10 ml Atom Lysepuffer (10 mM Tris (pH 8,0), 1 mM EDTA, 0,5 M NaCl, 1% Triton X-100, 0,5% Natriumdesoxycholat, 0,5% Lauroylsarkosin und 1x Proteaseinhibitoren). Inkubieren auf Eis für 10 min und Vortex alle 2-3 min. </li><li> Zentrifugieren Sie die Probe (830 × g bei 4 ° C für 8 min) und den Überstand verwerfen sorgfältig. </li><li> In 10 ml PBS Waschen des Pellets. Das Pellet (Chromatin-Anteil) kann bei -80 ° C nach der sofortigen Einfrieren in flüssigem Stickstoff gelagert werden. </li></ol> 5. Beschallung Chromatin (pro 2 x 10 7 Zellen) <ol><li> Hängen Sie das Chromatin Fraktion in 800 &amp; mgr; lmodifizierte Lysepuffer 3 (10 mM Tris (pH 8,0), 1 mM EDTA, 150 mM NaCl, 0,1% Natriumdeoxycholat, 0,1% SDS und Proteaseinhibitoren 1x) aufnehmen und in ein 1,5 ml Röhrchen. </li><li> Beschallen das Chromatin mit einem Ultraschallgerät (siehe Materialien), und die folgenden Bedingungen: Ausgang: 3; Pflicht: 100% (Dauerbetrieb); und Zeit: kostenlos. Zuführen 10-18 Zyklen der Ultraschallbehandlung für 10 s und Abkühlen auf Eis für 20 Sekunden. Eine zu starke Erwärmung zu vermeiden, läßt man die Proben auf Eis für 2 Minuten alle 5-6 Zyklen. Schaumbildung zu vermeiden, halten die Position der Spitze des Beschallungssonde 0,5 cm über dem Boden des Röhrchens. </li><li> Zentrifuge die Probe bei (16000 × g bei 4 ° C für 10 min) und den Überstand in ein 1,5 ml Röhrchen. Die beschallt Chromatin kann bei -80 ° C nach der sofortigen Einfrieren in flüssigem Stickstoff gelagert werden. </li></ol> 6. Auswertung der Chromatin Fragmentation <ol><li> Mischen Sie 10 ul der fragmentierten Chromatin mit 85 ul destilliertem Wasser. </li><li> In 4 ul5 M NaCl und Inkubation bei 65 ° CO / N. </li><li> 1 ul 10 mg / ml RNase A und bei 37 ° C für 45 min. </li><li> Add 2 ul 0,5 M EDTA (pH 8,0), 4 ul 1 M Tris (pH 6,8) und 1 ul 20 mg / ml Proteinase K, und dann bei 45 ° C inkubieren, für 1,5 Std. </li><li> Separate 10 ul der Probe durch Elektrophorese in einem 1% Agarose-Gel, das keine Verfärbung Farbstoffe wie SYBR Green enthält. </li><li> Fleck des Gels, um die Verteilung der Längen der fragmentierten Chromatin bewerten. Bedingungen, die eine mittlere Länge von 0,5 bis 2 kbp (Bereich von 0,2 bis 4 kbp) zu erzeugen, sind zu empfehlen. </li><li> Reinigen DNA von den verbleibenden Proben durch Phenol-Chloroform-Extraktion oder mit Hilfe eines DNA-Extraktions-Kits (siehe Materialien). Die gereinigte DNA kann als Eingabe-DNA verwendet werden, um die Ausbeuten an enChIP Analysen (siehe 8.11) zu schätzen. </li></ol> 7. Herstellung von Dynabeads Conjugated Antibodies mit (je 2 x 10 7 Zellen) <ol><li> Prepare zwei 1,5-ml-Röhrchen, eine für Pre-Clearing mit normalem Maus-IgG und die andere für die Inkubation mit dem Anti-FLAG-Ab. Zugabe von 150 &amp; mgr; l Protein G-konjugierten magnetischen Beads (siehe Materialien) in jedes Röhrchen. </li><li> Die Röhrchen auf einem Magneten stehen und warten für 3 min. Überstand verwerfen durch Pipettieren. </li><li> Aussetzung der Kügelchen in 1 ml PBS, enthaltend 0,01% Tween-20 (PBS-T). Die Röhrchen auf einem Magnetständer und warten Sie 2 min. Überstand verwerfen durch Pipettieren, und wiederholen Sie den Schritt. </li><li> Aussetzung der Kügelchen in 1 ml PBS-T, enthaltend 0,1% BSA. </li><li> In 15 &amp; mgr; g normalem Maus-IgG oder anti-FLAG Ab und drehen bei 4 ° CO / N. </li><li> Spin down kurz, dann legen Sie die Rohre auf einem Magneten stehen und warten für 3 min. Überstand verwerfen durch Pipettieren. </li><li> Suspend die Perlen in 1 ml PBS-T. Kehren Sie die Probe mehrmals und Spin-down kurz. Die Röhrchen auf einem Magneten stehen und warten für 3 Minuten, dann den Überstand verwerfen durch Pipettieren. Wiederholen Sie den Waschzwei weitere Male mit PBS-T (insgesamt drei Waschschritte). </li></ol> 8. Chromatin Immunopräzipitation (je 2 x 10 7 Zellen) <ol><li> Mischen das fragmentierte Chromatin in 5.3 hergestellten) (etwa 800 ul) mit einem Fünftel des Volumens (etwa 200 ul) von modifizierten Lysepuffer 3, enthaltend 5% Triton X-100 (Endkonzentration 1% Triton X-100). </li><li> Fügen Sie die Chromatin-Lösung für das Rohr, in dem Protein G-konjugierten mit normalem Maus-IgG, konjugiert magnetischen Kügelchen wurden hergestellt. Rotieren bei 4 ° C für 1 Stunde. </li><li> Das Röhrchen auf einem Magneten stehen und warten für 3 min. </li><li> Den Überstand in das Rohr, in dem G-Protein konjugiert mit anti-FLAG Ab konjugierten magnetischen Beads wurden hergestellt. Rotieren bei 4 ° CO / N. </li><li> Das Röhrchen auf einem Magneten stehen und warten für 3 min. Überstand verwerfen durch Pipettieren. </li><li> Aussetzung der Kügelchen in 1 ml salzarme Puffer (20 mM Tris (pH 8,0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0,1% SDS und Proteaseinhibitoren 1x) und drehen sich bei 4 ° C für 5 min. Das Röhrchen auf einem Magneten stehen und warten für 3 min. Überstand verwerfen durch Pipettieren und wiederholen Sie den Waschschritt. </li><li> Der Kügelchen mit Hochsalzpuffer (20 mM Tris (pH 8,0), 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0,1% SDS und Proteaseinhibitoren 1x) zweimal zu waschen. </li><li> Der Kügelchen mit LiCl-Puffer (10 mM Tris (pH 8,0), 1 mM EDTA, 250 mM LiCl, 0,5% IGEPAL CA-630, 0,5% Natriumdesoxycholat und 1x Proteaseinhibitoren) zweimal zu waschen. </li><li> Der Kügelchen mit TBS-IGEPAL CA-630 (50 mM Tris (pH 7,5), 150 mM NaCl, 0,1% IGEPAL CA-630 und 1x Proteaseinhibitoren) einmal zu waschen. </li><li> Suspendieren die Kügelchen in 200 &amp; mgr; l Elutionspuffer (50 mM Tris (pH 7,5), 150 mM NaCl, 0,1% IGEPAL CA-630, 1x Proteaseinhibitoren und 500 ug / ml 3 × FLAG-Peptid) und bei 37 ° C 20 min. Das Röhrchen auf einem Magneten stehen und warten für 3 min. Den Überstand in ein neues 1,5-ml-Tube und wiederholen Sie den ElutIonenschritt. </li><li> Reinigen DNA von kleiner Teil (zB 5%) des Eluats durch Phenol-Chloroform-Extraktion oder mit Hilfe eines DNA-Extraktions-Kits (siehe Materialien). Die gereinigte DNA kann für die PCR verwendet werden, mit spezifischen Primer-Sets, um die Erträge zu schätzen von enChIP Analysen durch Vergleich mit in Schritt 6.7, hergestellt wie zuvor 14,16 beschriebenen Eingangs DNA. </li></ol> 9. SDS-PAGE, Färbung und Massenspektrometrische Analyse <ol><li> Mischen das Eluat (400 ul) mit 1 ml 2-Propanol wurden 50 ul 3 M Natriumacetat (pH 5,2) und 5 ul 20 mg / ml Glykogen. Fällt das Chromatin bei -20 ° CO / N. </li><li> Zentrifugieren Sie die Probe (16.000 × g bei 4 ° C für 30 Minuten) und den Überstand verwerfen. Spülen Sie das Pellet mit 1 ml 70% Ethanol und erneut abzentrifugiert (16000 × g bei 4 ° C für 10 min). Überstand verwerfen vollständig durch Pipettieren. </li><li> Aussetzen des Pellets in 40 ul 2x Probenpuffer (125 mM Tris (pH 6,8), 10% 2-mercaptoethanol, 4% SDS, 10% Saccharose und 0,004% Bromphenolblau). Wirbel für 5 Minuten, um das Pellet vollständig aufzulösen, und dann bei 100 ° C für 30 min inkubiert, um die Proteine ​​zu denaturieren, und umgekehrt die Vernetzung. </li><li> Für SDS-PAGE, führen 40 ul der Probe auf das Gel, bis der Farbstoff erreicht 1 cm unterhalb der Bohrung. Hinweis: Wir verwenden normalerweise 4-20% Gradienten-Gele, aber andere% Gele verwendet werden. </li><li> Fleck das Gel mit Coomassie Brilliant Blue oder Silberfärbung. </li><li> Schneiden Sie das Gel in fünf Stücke (2 mm Höhe). </li><li> Führen Sie in Gel-Verdauung und massenspektrometrische Analyse wie zuvor 13-16 beschrieben. </li><li> Für SILAC Experimente mit 5 x 10 7 Zellen jeweils (insgesamt 1 × 10 8 Zellen), auszusetzen Pellet aus den ersten fünf Rohre in 40 ul 2x Probenpuffer (es ist nicht notwendig zu skalieren). </li></ol> 10. Reinigung von RNA und RNA-Seq Analysis <ol><li> Um die RNA nach enChIP reinigen, fügen Sie 5 U / ml RNase Inhibitor (see Materials), um alle Pufferlösungen, ausgenommen modifizierte Lysepuffer 3 und Elutionspuffer, an die 40 U / ml RNase-Inhibitor hinzuzufügen. </li><li> Mischen das Eluat (400 ul) mit 16 ul 5 M NaCl und Inkubation bei 65 ° C für 2 Std. </li><li> 1 ml säure Guanidinium-Phenol-Reagens (siehe Materialien) zur Probe. Vortex für 15 Sekunden und dann Inkubation bei Raumtemperatur für 5-15 min. Zentrifugieren Sie die Probe für 15 min bei 12000 g und RT. </li><li> Den Überstand in ein neues 1,5-ml-Röhrchen und fügen Sie 5 ul p-Bromanisol. Vortex für 15 Sekunden und dann Inkubation bei Raumtemperatur für 3-5 min. Zentrifugieren Sie die Probe für 10 min bei 12000 g und RT. </li><li> Den Überstand (etwa 1 ml) in ein neues 2-ml-Tube und 1 ml 2-Propanol. Das Röhrchen und Inkubation bei Raumtemperatur für 10 min. Laden Sie die Mischung auf eine Spalte in einer RNA-Reinigungskits (siehe Materialien). Zentrifugieren Sie die Säule für 1 min bei 12000 g und RT. </li><li> Waschendie Säule wurde mit 400 ul Waschpuffer RNA in einem RNA-Reinigungs-Kits (siehe Materialien). </li><li> Werden 80 &amp; mgr; l DNase I-Cocktail (Mischung von 5 ul DNase I (1 U / ul), 8 &amp; mgr; l von 10 × DNase I-Reaktionspuffer, 3 ul DNase / RNase-freies Wasser und 64 ul RNA Waschpuffer eine RNA-Reinigungs-Kits (siehe Materialien)) auf die Säule. Inkubieren der Probe bei 37 ° C für 15 min und dann zu zentrifugieren für 30 Sekunden bei 12.000 × g und RT. </li><li> Mit 400 &amp; mgr; l RNA PreWash Puffer in einer RNA-Reinigungs-Kits (siehe Materialien), wasche zweimal die Spalte. </li><li> Elution der RNA mit 50 ul DNase / RNase-freies Wasser. Die eluierte RNA zur RNA-Sequenzierung verwendet werden. </li></ol>

<ol>
	<li>Zhang, X. Y., Horz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+highly+purified+satellite+DNA+containing+chromatin+from+the+mouse.">Analysis of highly purified satellite DNA containing chromatin from the mouse.</a> Nucleic Acids Res. 10, 1481-1494 (1982).</li><li>Workman, J. L., Langmore, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoprotein+hybridization:+a+method+for+isolating+specific+genes+as+high+molecular+weight+chromatin.">Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin.</a> Biochemstry. 24, 7486-7497 (1985).</li><li>Boffa, L. C., Carpaneto, E. M., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+active+genes+containing+CAG+repeats+by+DNA+strand+invasion+by+a+peptide+nucleic+acid.">Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid.</a> Proc. Natl. Acad. Sci. USA. 92, 1901-1905 (1995).</li><li>Jaskinskas, A., Hamkalo, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+initial+characterization+of+primate+satellite+chromatin.">Purification and initial characterization of primate satellite chromatin.</a> Chromosome Res. 7, 341-354 (1999).</li><li>Griesenbeck, J., Boeger, H., Strattan, J. S., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+specific+chromatin+segments+from+chromosomal+loci+in+yeast.">Affinity purification of specific chromatin segments from chromosomal loci in yeast.</a> Mol. Cell Biol. 23, 9275-9282 (2003).</li><li>Ghirlando, R., Felsenfeld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+studies+on+defined+heterochromatin+fragments+support+a+30-&#181;m+fiber+having+six+nucleosomes+per+turn.">Hydrodynamic studies on defined heterochromatin fragments support a 30-&#181;m fiber having six nucleosomes per turn.</a> J. Mol. Biol. 376, 1417-1425 (2008).</li><li>D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins+associated+with+specific+genomic+loci.">Purification of proteins associated with specific genomic loci.</a> Cell. 136, 175-186 (2009).</li><li>Hoshino, A., 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=Insertional+chromatin+immunoprecipitation:+a+method+for+isolating+specific+genomic+regions.">Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions.</a> J. Biosci. Bioeng. 108, 446-449 (2009).</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=Direct+idenification+of+insulator+components+by+insertional+chromatin+immunoprecipitation.">Direct idenification of insulator components by insertional chromatin immunoprecipitation.</a> PLoS One. 6, e26109 (2011).</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+by+insertional+chromatin+immunoprecipitation+(iChIP)+with+a+second-generation+tagged+LexA+DNA-binding+domain.">Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain.</a> Adv. Biosci. 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.

Die Identifizierung von Molekülen mit spezifischen genomischen Regionen von Interesse verbunden ist erforderlich, um die Mechanismen der Regulation von genomischer Funktionen wie Transkription und epigenetischen Regulation verstehen. Obwohl mehrere Techniken für die biochemische Analyse von spezifischen genomischen Regionen 1-7 entwickelt wurden, werden sie nicht in großem Umfang zu diesem Zeitpunkt wegen ihrer inhärenten Probleme wie begrenzte Anwendung (beispielsweise nur für eine hohe Kopienzahl Loci oder Loci mit repeats) und gebrauchte zu viel Zeit und Anstrengungen erforderlich. Um biochemische Analyse spezifischer Genomregionen leicht durchzuführen, haben wir zwei locusspezifische Chromatinimmunpräzipitation (Chip) Technologien, nämlich Insertions- Chip (Ichip) 8-13 und ausgeführt, DNA-bindendes Molekül vermittelte Chip (enChIP) entwickelt 14-17 . In Ichip wird eine Locus von Interesse durch Insertion Erkennungssequenzen eines exogenen DNA-bindendes Protein wie Lex taggedA. Die Ortskurve wird dann durch Affinitätsreinigung unter Verwendung der markierten DNA-bindenden Protein isoliert. In enChIP, konstruiert DNA-bindende Moleküle, wie Zinkfinger-Proteinen, Transkriptionsaktivator-like (TAL) Proteine ​​und gruppierten regelmäßig beabstandeten kurzen palindromischen Repeats (CRISPR) -Komplexe verwendet werden, um einen Ort von Interesse zu markieren (Abbildung 1). Anschließend wird die Genomregion durch Affinitätsreinigung des markierten DNA-bindenden Moleküle isoliert. Einer der Vorteile der enChIP über Ichip dass Insertion Erkennungssequenzen eines exogenen DNA-bindendes Protein ist nicht notwendig. Targeting von Loci unter Verwendung CRISPR-Komplexe, bestehend aus einer katalytisch inaktiven Form Cas9 (dCas9) und einer Führungs RNA (gRNA) ist viel einfacher als Targeting dieser Regionen durch Ichip oder enChIP Verwendung TAL und Zinkfinger-Proteine. Hier beschreiben wir einen Schritt-für-Schritt-Protokoll für enChIP kombiniert mit Massenspektrometrie und RNA-Sequenzierung (RNA-Seq) locus-Assoziationen zu identifizierented Proteinen und RNAs, die jeweils.

<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.

Insgesamt kann 1-30% des Ziels genomischen Regionen mittels enChIP gereinigt werden. Figur 3 enthält repräsentative Daten, die die Ausbeuten an enChIP Analysen Targeting Telomeren und den Promotor der Interferon (IFN) regulatory factor-1 (IRF-1) -Gen. Als Beispiele für typische Ergebnisse, Tabelle 1 sind die Proteine ​​mit der IRF-1-Promotors in einer IFN-spezifischen Weise durch enChIP-SILAC Tabelle identifiziert zugeordnet 2 listet die Telomer-bindenden Proteinen durch enChIP kombiniert mit Massenspektrometrie identifiziert (enChIP-MS) und Tabelle 3 zeigt die RNAs mit Telomeren enChIP-RNA-Seq identifiziert verbunden. <img alt="Abbildung 1" src="/files/ftp_upload/53478/53478fig1.jpg" /> Abbildung 1. Übersicht über enChIP. enChIP Verwendung CRISPR (A, B) und Elemente (C, D, E </strong&gt;) angezeigt. Ein Ort von Interesse in Verbindung mit technisch DNA-bindende Moleküle wie einen TAL Proteins oder des CRISPR-System, bestehend aus einer katalytisch inaktiven Form Cas9 (DCAS) markiert und Führungs RNA (gRNA). Die molekularen Wechselwirkungen mit Formaldehyd oder anderen Vernetzern, gegebenenfalls fixiert. Anschließend fixiert Chromatin durch Beschallung oder enzymatische Verdauung fragmentiert. Die markierten genomischen Regionen werden durch Affinitätsreinigung gereinigt. Schließlich wird die Vernetzung umgekehrt, und wechselwirkenden Molekülen (genomischen Regionen, RNAs und Proteine) werden unter Verwendung der nächsten Generation Sequenzierung und Massenspektrometrie identifiziert. <a href="https://www.jove.com/files/ftp_upload/53478/53478fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53478/53478fig2.jpg" /> Abbildung 2. Reihenfolge der gBlock. Die U6-Promotor (in schwarz), die G-Nukleotid-bevor die Führungssequenz (in blau), die Führungssequenz (gRNA Spacer) (in rot), das Gerüst-Sequenz (in grün) und die Terminator-Sequenz (in orange) angezeigt. <img alt="Figur 3" src="/files/ftp_upload/53478/53478fig3.jpg" /> Abbildung 3. Die Renditen von repräsentativen enChIP Analysen. (A) Prozentuale Eingänge für das Soll-IRF-1-Locus und dem Nicht-Ziel Sox2 Locus. (B) Prozentsatz Eingänge für die Ziel Telomere und Nichtziel-γ-Satelliten. Die Zahlen wurden angepasst und aus früheren Veröffentlichungen 15,16 modifiziert. <table fo:keep-together.within-page="1"><tbody><tr><td> Kategorien </td><td> Proteine </td></tr><tr><td> Transkription </td><td> DDX1, PARP1, CKAP4, Pescadillo Homolog, PURβ, aktiviert RNA Polymerase II Transkriptionsaktivator p15, BTF3, Myb-bindendeProtein 1A </td></tr><tr><td> Histondeacetylierung, Corepressor Komponenten </td><td> RBBP4, PA2G4, TBL3 </td></tr><tr><td> Acetyltransferase </td><td> Protein-Arginin-N-Methyltransferase 1 </td></tr><tr><td> DNA-Topoisomerase </td><td> DNA-Topoisomerase 2α </td></tr><tr><td> Histone </td><td> Histone H2A.Z, Histon H3.2 </td></tr></tbody></table> Tabelle 1:. Beispiele für Proteine, die mit dem menschlichen IRF-1-Promotor-Region in einem IFN-spezifischen Weise durch enChIP-SILAC identifizierten zugehörigen Die Tabelle ist aus einer früheren Veröffentlichung 16 angepasst. <table fo:keep-together.within-page="1"><tbody><tr><td> Kategorien </td><td> Proteine </td></tr><tr><td> Säugetier Telomer-bindenden Proteinen </td><td> PML, RPA, CDK1, PARP1, PCBP1 </td></tr><tr><td> Telomer-binden Proteine ​​in Hefe oder anderen Organismen </td><td> IMAP4 </td></tr><tr><td> Proteine ​​interagieren mit Telomer-Binde Proteinen assoziiert [Telomer-bindenden Protein] </td><td> DNA-Polymerase α (POLA1) [Cdc13p], ARMC6 [TRF2], CtBP1 [FoxP2-POT1], Exportin-5 [TERT], GNL3L [TRF1], Exportin-1 [TERT], 14-3-3 [TERT] </td></tr><tr><td> Proteine ​​zu lokalisieren, um Heterochromatin </td><td> BEND3 </td></tr><tr><td> Proteine ​​regulieren epigenetische Markierungen </td><td> KDM5C </td></tr><tr><td> Proteine, deren Mutationen betreffen Telomerfunktion </td><td> DNA-Polymerase α (POLA1), HAT1, Nup133, CDK7, DPOE1, PRDX1, TYSY, Glutamat-Cystein-Ligase Glutaredoxin SMRC1 </td></tr></tbody></table> Tabelle 2:. Beispiele für Proteine ​​mit Maus Telomeren enChIP-MS identifiziert, assoziiert Tabelle bereits adapted aus einer früheren Veröffentlichung 15. <table fo:keep-together.within-page="1"><tbody><tr><td> Kategorien </td><td> RNAs </td></tr><tr><td> Telomerase-Komponenten </td><td> Terc, rMRP </td></tr><tr><td> Telomer-RNAs </td><td> Terras </td></tr><tr><td> scaRNAs </td><td> Scarna6, Scarna10, Scarna13, Scarna2 </td></tr><tr><td> H / ACA snoRNAs </td><td> Snora23, Snora74a, Snora73b, Snora73a </td></tr><tr><td> C / D snoRNAs </td><td> Snord17, Snord15a, Snord118 </td></tr><tr><td> lncRNA </td><td> Neat1 </td></tr></tbody></table> Tabelle 3: Beispiele für RNAs mit der Maus Telomere durch enChIP-RNA-Seq Die Tabelle hat sich von einer früheren Veröffentlichung 17 angepasst wurden identifiziert verbunden..

Watch this Scientific Journal Video about Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP at JoVE.com

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

Isolierung von spezifischen genomischen Regionen und Identifizierung von assoziierten Moleküle durch 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. 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

Research

JoVE Journal

Biology

10.4K Aufrufe.  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.

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

Isolation of Genomic DNA from Mouse Tails

Isolierung genomischer DNA aus Maus-Tails

Forschung

Biologie

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.

Competitive Genomic Screens von Barcoded Hefe Bibliotheken

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.

Identifizierung und Charakterisierung von Protein-Glykosylierung mit speziellen Endo-und Exoglycosidasen

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.

Isolation von Normal und Krebs-assoziierte Fibroblasten von Fresh Gewebe durch 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 ...

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.

Das Utility von Stage-spezifischen Mid-zu-spät<em&gt; Drosophila</em&gt; Follikel Isolation

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.

Isolierung von mRNAs Verbunden mit Hefe-Mitochondrien Study Mechanismen der lokalisierten Übersetzung

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.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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.

Entfernen von exogenen Materialien aus dem äußeren Teil der gefrorenen Kerne der Antike biologische Gemeinschaften beherbergte Innen zur Untersuchung

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.

Pflanzenanalysen Promoter: Identifizierung und Charakterisierung der Root Knoten bestimmten Promoter in die Gartenbohne

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.

Isolierung und Bildgebung von live, intakten Herzschrittmacherregionen des Nierenbeckens der Maus durch Vibratom-Schnitte

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