Wir danken Dr. Yoko Nabeshima an der Kyoto University für ihren Unterricht auf der Dissektionstechnik und Frau Aki Miyakawa bei Fujita Health der Universität für ihre Unterstützung zu filmen. Von MEXT in Japan, und durch - Diese Arbeit wurde durch das Programm zur Förderung der Grundrechte Studies in Health Sciences der National Institute of Biomedical Innovation, ein Grant-in-Aid for Scientific Research auf Schwerpunktbereiche-Integrative Hirnforschung (Shien) unterstützt Grant-in-Aid von CREST der Japan Science and Technology Agency.

Dissection der hippocampalen <ol><li> In einer tief narkotisierten Maus, sorgfältig sezieren das Gehirn aus dem Schädel und legen Sie sie in eiskaltes Phosphat-gepufferter Salzlösung (PBS). </li><li> In eine Petrischale mit eiskaltem PBS, schneiden das Gehirn entlang der Längsfissur des Großhirns mit einem chirurgischen Messer und schnitt den Regionen hinter Lambda (Mittelhirn, Hinterhirn und Kleinhirn). </li><li> Legen Sie die Gehirnhälfte medialen Seite und, mit einer Pinzette vorsichtig entfernen das Zwischenhirn (Thalamus und Hypothalamus) im Rahmen einer Dissektionsmikroskop. Dies legt die mediale Seite des Hippocampus, so dass für die Visualisierung des Gyrus dentatus. Der Gyrus dentatus ist zu unterscheiden von Ammonshorn durch die Lücken zwischen ihnen. Eine Verletzung des Hippocampus oder Umgebung wird es immer schwieriger, die Gyrus dentatus zu isolieren. </li><li> Legen Sie eine scharfe Nadelspitze (zB 27-Gauge-Nadel) in jeder Seite des Gyrus dentatus (Grenzen des Gyrus dentatus und Ammonshorn; Abbildung 1), und schieben Sie die Nadeln nur oberflächlich entlang der Septo-zeitliche Achse des Hippocampus zu isolieren Gyrus dentatus. </li><li> Nehmen Sie den isolierten Gyrus dentatus mit einer Nadel oder einer Pinzette und legen Sie sie in ein Probenröhrchen. Die so erhaltenen Gyrus dentatus Gewebeprobe kann ab sofort für jeden Assay verwendet werden oder in eine Tiefkühltruhe für den späteren Gebrauch. </li><li> Isolieren Sie die Gyrus dentatus von der anderen Gehirnhälfte mit der gleichen Methode. </li></ol> Quantitative real-time PCR Der Gyrus dentatus wurde unter Verwendung der oben genannten Methode und die übrigen Hippocampus wurde als Ammonshorn Probe von Wildtyp-Mäusen seziert. Real-time PCR von beta-Aktin, TDO2, DSP, Mrg1b und Tyro3 wurden mit dem Gyrus dentatus und der Ammonshorn Proben wie zuvor beschrieben 1 durchgeführt. Primer 5&#39;-CTGGCGAGATCACGATGACG und 5&#39;-AAGCTACGCTGTTGTCTAACC wurden Mrg1b und GCCTCCAAATTGCCCGTCA und 5&#39;-CCAGCACTGGTACATGAGATCA für Tyro3 verwendet. Microarray-Analyse (- Mäuse alpha-CaMKII + /), wie beschrieben bisher 1 Microarray-Experimente wurden mit männlichen Wildtyp-Mäusen und Mäusen, die heterozygot für die alpha-Isoform der Calcium / Calmodulin-abhängige Proteinkinase II durchgeführt. Kurz gesagt, wurde RNA aus dem gesamten Hippocampus oder Gyrus dentatus von Wildtyp-und mutierten Mäusen isoliert mit einem Mouse Genome 430 2,0 Array (Affymetrix, Santa Clara, CA) hybridisiert, und jeder GeneChip wurde durch eine Affymetrix GeneChip Scanner 3000 (GCS3000) gescannt . GeneChip Analyse wurde mit Microarray Analysis Suite Version 5.0 durchgeführt.

<ol>
	<li>Yamasaki, N., Maekawa, M., Kobayashi, K., Kajii, Y., Maeda, J., Soma, M., Takao, K., Tanda, K., Ohira, K., Toyama, K., Kanzaki, K., Fukunaga, K., Sudo, Y., Ichinose, H., Ikeda, M., Iwata, N., Ozaki, N., Suzuki, H., Higuchi, M., Suhara, T., Yuasa, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-CaMKII+deficiency+causes+immature+dentate+gyrus,+a+novel+candidate+endophenotype+of+psychiatric+disorders.">Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.</a> Mol. Brain. 1, 6-6 (2008).</li><li>Insausti, A. M., Megias, M., Crespo, D., Cruz-Orive, L. M., Dierssen, M., Vallina, I. F., Insausti, R., Florez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+neuronal+number+in+Ts65Dn+mice:+a+murine+model+of+Down+syndrome.">Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of Down syndrome.</a> Neurosci. Lett. 253, 175-175 (1998).</li><li>Redwine, J. M., Kosofsky, B., Jacobs, R. E., Games, D., Reilly, J. F., Morrison, J. H., Young, W. G., Bloom, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentate+gyrus+volume+is+reduced+before+onset+of+plaque+formation+in+PDAPP+mice:+a+magnetic+resonance+microscopy+and+stereologic+analysis.">Dentate gyrus volume is reduced before onset of plaque formation in PDAPP mice: a magnetic resonance microscopy and stereologic analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 1381-1381 (2003).</li><li>Lein, E. S., Zhao, X., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+molecular+atlas+of+the+hippocampus+using+DNA+microarrays+and+high-throughput+in+situ+hybridization.">Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization.</a> J. Neurosci. 24, 3879-3879 (2004).</li><li>Matsuo, N., Yamasaki, N., Ohira, K., Takao, K., Toyama, K., Eguchi, M., Yamaguchi, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+changes+underlying+the+working+memory+deficit+in+alpha-CaMKII+heterozygous+knockout+mice.">Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice.</a> Front. Behav. Neurosci. 3, 20-20 (2009).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

Tierversuche wurden in Übereinstimmung mit dem National Institute of Health Guide für Pflege und Verwendung von Labortieren durchgeführt, und von der Pflege der Tiere und die Nutzung Ausschuss bei Fujita Health University.

Der Gyrus dentatus belegt ca. 25% bis 30% des Volumens des Hippokampus 2,3. Es hat eine einzigartige Zellzusammensetzung und spielt eine entscheidende Rolle in verschiedenen Funktionen des Gehirns. Daher sind Techniken, um die Gyrus dentatus zu isolieren für die Analyse der Ereignisse, die speziell treten in dieser Region. Hier haben wir gezeigt, ein Verfahren zur effizienten sezieren Gyrus dentatus aus adulten Maus Hippocampus und bestätigt die Genauigkeit der Technik. Zunächst zeigte histologische Untersuchung, dass der Gyrus dentatus ohne Verunreinigung durch andere Regionen (Abbildung 1) wurde abgetrennt, was darauf hinweist, dass eine reine Gyrus dentatus Probe vorbereitet werden können. Zweitens bestätigten wir, dass die präparierten Gewebe Gyrus dentatus ist durch die Durchführung real-time PCR des Gyrus dentatus-spezifische Gene, TDO2 und DSP-und Ammonshorn bereichert Gene, Mrg1b und Tyro3 4 (Abbildung 2). Die mRNA Expression von TDO2 (p = 0,000023; n ist = 4 bzw. 4) und DSP (p = 0,0000030; n ist = 4 bzw. 4) im Gyrus dentatus Proben wurden bei offensichtlich höhere erkannt, während Mrg1b (p = 0.000080; n ist = 4 bzw. 4) und Tyro3 (p = 0,00017; n ist = 4 bzw. 4) wurden niedrigere, als die im Ammonshorn Proben. Beta-Aktin-Expression nicht in diesen Proben unterscheiden (p = 0,11; n ist = 4 und 4, jeweils). So können wir überprüfen, ob der Gyrus dentatus genau wurde von der Durchführung solcher einfachen Echtzeit-PCR-Experimente seziert. Drittens, um den Nutzen dieser Methode Dissektion beurteilen, haben wir die mRNA Expression von ganzen Hippocampus mit dem Gyrus dentatus. Whole Hippocampus und Gyrus dentatus von Wildtyp-(n ist = 9 bzw. 4) und alpha-CaMKII + / erhalten - Mäuse (n ist = 18 bzw. 4) wurden für die Microarray-Analyse aufbereitet und für alle Gene hat, die fach- Veränderung wurde durch Division der Mutante Wert durch die wild-type-Wert berechnet. Die Ergebnisse zeigten, dass die Veränderungen in mRNA-Expression, insbesondere des Gyrus dentatus-spezifische Moleküle wie DSP und TDO2, mit wurden bis zu einem 5-fache Steigerung der Empfindlichkeit in Gyrus dentatus Proben im Vergleich zum gesamten Hippocampus-Proben (Tabelle 1) festgestellt. Wir haben bereits gezeigt, dass alpha-CaMKII + / - Mäuse zeigen Verhaltensweisen der menschlichen psychiatrischen Störungen wie Arbeitsspeicher Defizite und eine übertriebene infradian Rhythmus 1,5 verwandt. Darüber hinaus sind morphologische und elektrophysiologische Eigenschaften des Gyrus dentatus Neuronen in mutierten Mäusen ähneln denen von unreifen Gyrus dentatus Nervenzellen im normalen Nagern, was darauf hinweist, dass die Neuronen in diesen mutierten Mäusen bis zur Endfälligkeit zu 1 zu entwickeln scheitern. Die unreifen Gyrus dentatus und down-regulierten Expression von DSP und TDO2 mRNA in alpha-CaMKII + / - Mäuse sind im Einklang mit dem Befund, dass DSP und TDO2 als Marker für reife Körnerzellen kann im Gyrus dentatus (Ohira et al verwendet werden, nicht veröffentlicht. Daten). Zusammengenommen können diese bequeme und genaue Dissektionstechnik zuverlässig für Studien über den Gyrus dentatus konzentriert eingesetzt werden. Gyrus dentatus Gewebe unter Verwendung dieser Methode ist anwendbar auf andere Arten von Analysen sowie, einschließlich Proteomics und Zellbiologie Analysen. <img src="/files/ftp_upload/1543/1543fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Verification der isolierten Gyrus dentatus durch die histologische Untersuchung. A Frontalschnitt des Gehirns nach Trennung Gyrus dentatus wurde für Nissl-Färbung (links) verarbeitet, und eine schematische Darstellung von Gehirn der Maus atlas6 angepasst repräsentiert die etwa auf dem Niveau des Abschnitts in der linken Seite (rechts) gezeigt. Die Pfeile zeigen die Richtung der Nadelspitze Insertion. Scale-bar, 1 mm. <img src="/files/ftp_upload/1543/1543fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Nachweis der isolierten Gyrus dentatus durch real-time PCR. Der Gyrus dentatus und der Ammon s Horn von vier Wildtyp-Mäusen gewonnen wurden real-time PCR von beta-Aktin, TDO2, DSP, Mrg1b und Tyro3 verarbeitet. Die Ergebnisse werden präsentiert als Mittelwert ± SEM. Für die statistische Analyse wurde Student s t-Test eingesetzt, und p-Werte eingehalten werden: beta-Aktin, p = 0,11; TDO2, p = 0,000023 (** 1); Dsp, p = 0,0000030 (** 2); Mrg1b, p = 0.000080 (** 3); und Tyro3, ​​p = 0,00017 (** 4). Tabelle 1. . Microarray-Analyse der gesamten Hippocampus und Gyrus dentatus Gene differentiell in Gyrus dentatus und ganze Hippocampus von alpha-CaMKII ausgedrückt + / - Mäuse wurden durch die Berechnung der fold-change aus, dass in Wildtyp-Mäusen nachgewiesen bestimmt. Mäuse - Die Daten wurden auf statistische Signifikanz mit dem Student s t-Test zwischen Wildtyp-und alpha-CaMKII + / analysiert. Unter den Genen, deren Expression zeigte p &lt;0,05 inGyrus dentatus von alpha-CaMKII + / - Mäusen im Vergleich zu der Wildtyp-Mäusen sind die Top-50 Gene aufgelistet. Beachten Sie, dass die Zahl von Proben für Gyrus dentatus viel weniger als jene für die ganze Hippocampus sind. AffyID, Affymetrix-Sonde Kennung; CKII, alpha-CaMKII + / - Mäuse, WT, Wildtyp-Mäusen. <table border="1"><tbody><tr><td></td><td></td><td></td><td></td><td colspan="2"> Gyrus dentatus (p &lt;0,05) WT: n = 4, CKII + / -: n = 4 </td><td></td><td colspan="2"> Whole Hippocampus WT: n = 9, CKII + / -: n = 18 </td></tr><tr><td> Gene Titel </td><td> Genbank </td><td> AffyID </td><td></td><td> Falten Sie ändern </td><td> p-Wert </td><td></td><td> Falten Sie ändern </td><td> p-Wert </td></tr><tr><td> Desmoplakin </td><td> AV297961 </td><td> 1435494_s_at </td><td></td><td> 0,011018913 </td><td> 7.02694E-06 </td><td></td><td> 0,037021003 </td><td> 1.86126E-13 </td></tr><tr><td> Desmoplakin </td><td> AV297961 </td><td> 1435493_at </td><td></td><td> 0,014369734 </td><td> 7.86747E-06 </td><td></td><td> 0.04232106 </td><td> 1.00579E-12 </td></tr><tr><td> Tryptophan 2,3-Dioxygenase </td><td> AI098840 </td><td> 1419093_at </td><td></td><td> 0,020986484 </td><td> 5.23546E-09 </td><td></td><td> 0,101037776 </td><td> 4.14823E-13 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452106_at </td><td></td><td> 0,075479901 </td><td> 1.05191E-08 </td><td></td><td> 0,234001154 </td><td> 1.66301E-15 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452107_s_at </td><td></td><td> 0,079457767 </td><td> 1.40433E-07 </td><td></td><td> 0,177974715 </td><td> 3.9758E-12 </td></tr><tr><td> Thyreotropin-Releasing-Hormon-Rezeptor- </td><td> M59811 </td><td> 1449571_at </td><td></td><td> 0,103105815 </td><td> 0,003093796 </td><td></td><td> 0,801412732 </td><td> 0,283994361 </td></tr><tr><td> Ryanodinrezeptor 1, Skelettmuskel </td><td> X83932 </td><td> 1427306_at </td><td></td><td> 0,104825517 </td><td> 3.38513E-07 </td><td></td><td> 0,650685017 </td><td> 0,000308462 </td></tr><tr><td> nescient Helix Loop Helix 1 </td><td> NM_010916 </td><td> 1419533_at </td><td></td><td> 9.431896 </td><td> 6.7979E-06 </td><td></td><td> 4,078815314 </td><td> 5.27E-11 </td></tr><tr><td> copine Familienmitglied IX </td><td> BB274531 </td><td> 1454653_at </td><td></td><td> 9.159157 </td><td> 7.99492E-06 </td><td></td><td> 1,797304153 </td><td> 0,000296375 </td></tr><tr><td> Doublecortin-like Kinase-3 </td><td> BB326709 </td><td> 1436532_at </td><td></td><td> 0,109336662 </td><td> 1.95278E-07 </td><td></td><td> 0.56697229 </td><td> 2.62633E-08 </td></tr><tr><td> Calpain 3 </td><td> AF127766 </td><td> 1426043_a_at </td><td></td><td> 0,111269769 </td><td> 8.07053E-06 </td><td></td><td> 0,370956608 </td><td> 2.04421E-14 </td></tr><tr><td> Erwachsene männliche corpus striatum cDNA, RIKEN in voller Länge angereichert Bibliothek, Klon: C030023B07 Produkt: unklassifizierbare, voll Insertsequenz </td><td> BB357628 </td><td> 1460043_at </td><td></td><td> 0,118712341 </td><td> 6.16926E-07 </td><td></td><td> 0,682339204 </td><td> 2.33001E-06 </td></tr><tr><td> Kollagen und Calciumbindevermögen EGF Domänen 1 </td><td> AV264768 </td><td> 1437385_at </td><td></td><td> 0,124043978 </td><td> 3.65669E-05 </td><td></td><td> 0,488394112 </td><td> 4.05538E-06 </td></tr><tr><td> Amyloid-beta (A4)-Precursor-Protein-Bindung, Familie A, Mitglied 2-Bindungsprotein </td><td> AK013520 </td><td> 1431946_a_at </td><td></td><td> 7.7986307 </td><td> 1.2098E-06 </td><td></td><td> 2,099164713 </td><td> 1.67047E-06 </td></tr><tr><td> Calbindin-28K </td><td> BB177770 </td><td> 1456934_at </td><td></td><td> 0,130255444 </td><td> 3.32186E-06 </td><td></td><td> 0,572605751 </td><td> 1.99157E-10 </td></tr><tr><td> Transkribiert locus </td><td> AV328597 </td><td> 1443322_at </td><td></td><td> 0,133290835 </td><td> 5.43583E-06 </td><td></td><td> 0,562767164 </td><td> 7.56544E-06 </td></tr><tr><td> Neuropeptid Y-Rezeptor Y2 </td><td> NM_008731 </td><td> 1417489_at </td><td></td><td> 0,135319609 </td><td> 0,000113407 </td><td></td><td> 0,781498474 </td><td> 0.00394504 </td></tr><tr><td> ras responsive element binding protein 1 </td><td> BE197381 </td><td> 1428657_at </td><td></td><td> 0,138235114 </td><td> 7.93691E-07 </td><td></td><td> 0,651220705 </td><td> 2.94209E-05 </td></tr><tr><td> Glia-Zelllinie neurotrophen Faktor Familie Rezeptor alpha 2 </td><td> BB284482 </td><td> 1433716_x_at </td><td></td><td> 0,139062563 </td><td> 2.35371E-06 </td><td></td><td> 0,669544709 </td><td> 0,000214146 </td></tr><tr><td> preproenkephalin 1 </td><td> M13227 </td><td> 1427038_at </td><td></td><td> 6.9850435 </td><td> 2.39074E-08 </td><td></td><td> 1,766018828 </td><td> 0,000250501 </td></tr><tr><td> RIKEN cDNA 1810010H24 Gen </td><td> BI729991 </td><td> 1428809_at </td><td></td><td> 6.8658915 </td><td> 1.88516E-05 </td><td></td><td> 2.77573142 </td><td> 6.81865E-09 </td></tr><tr><td> Ryanodinrezeptor 1, Skelettmuskel </td><td> BG793713 </td><td> 1457347_at </td><td></td><td> 0,151364292 </td><td> 3.35612E-05 </td><td></td><td> 0,503144617 </td><td> 4.32907E-05 </td></tr><tr><td> Protocadherin 21 </td><td> NM_130878 </td><td> 1418304_at </td><td></td><td> 0,152671849 </td><td> 8.57783E-06 </td><td></td><td> 0,670714726 </td><td> 1.56309E-05 </td></tr><tr><td> cornichon homolog 3 (Drosophila) </td><td> NM_028408 </td><td> 1419517_at </td><td></td><td> 0,153724144 </td><td> 8.90755E-06 </td><td></td><td> 0.95780695 </td><td> 0,661055608 </td></tr><tr><td> Harakiri, BCL2 interagierende Protein (enthält nur BH3-Domäne) </td><td> BQ175572 </td><td> 1439854_at </td><td></td><td> 0,154284407 </td><td> 2.0118E-05 </td><td></td><td> 0.56516812 </td><td> 4.86925E-09 </td></tr><tr><td> Kohlenhydrat-(N-Acetyl 4-0) Sulfotransferase 9 </td><td> AK017407 </td><td> 1431897_at </td><td></td><td> 0,155238951 </td><td> 5.37423E-06 </td><td></td><td> 1.14910007 </td><td> 0,215637733 </td></tr><tr><td> Calpain 3 </td><td> AI323605 </td><td> 1433681_x_at </td><td></td><td> 0,160871988 </td><td> 1.07655E-05 </td><td></td><td> 0,477164757 </td><td> 1.33753E-11 </td></tr><tr><td> Zink-Finger, CCHC Domäne mit 5 </td><td> BQ126004 </td><td> 1437355_at </td><td></td><td> 0,161812078 </td><td> 3.08262E-06 </td><td></td><td> 0,421252632 </td><td> 0.01152969 </td></tr><tr><td> Loricrin </td><td> NM_008508 </td><td> 1448745_s_at </td><td></td><td> 0,165129967 </td><td> 1.86362E-05 </td><td></td><td> 0,639733409 </td><td> 0,000729772 </td></tr><tr><td> spondin 1, (f-spondin) der extrazellulären Matrix Protein </td><td> BC020531 </td><td> 1451342_at </td><td></td><td> 0,168035879 </td><td> 6.67867E-07 </td><td></td><td> 0,821042412 </td><td> 0,023650765 </td></tr><tr><td> RIKEN cDNA A930035E12 Gen </td><td> AV348640 </td><td> 1429906_at </td><td></td><td> 5.9086795 </td><td> 1.747E-07 </td><td></td><td> 1,470383201 </td><td> 0,104085454 </td></tr></tbody><tbody><tr><td> BB247294 RIKEN in voller Länge angereichert, 7 Tage die Neugeborenen Kleinhirn Mus musculus cDNA-Klon A730018G18 3 &#39;, mRNA-Sequenz. </td><td> BB247294 </td><td> 1447907_x_at </td><td></td><td> 5.9047494 </td><td> 1.04931E-05 </td><td></td><td> 1,968147585 </td><td> 0.00010636 </td></tr><tr><td> FERM Domäne mit 3 </td><td> BB099015 </td><td> 1437075_at </td><td></td><td> 5.860216 </td><td> 0,000345581 </td><td></td><td> 2,780297178 </td><td> 1.83072E-06 </td></tr><tr><td&gt; Neuronalen Pentraxin 2 / / / hypothetisches Protein LOC100044234 </td><td> NM_016789 </td><td> 1420720_at </td><td></td><td> 5.7568517 </td><td> 1.34227E-06 </td><td></td><td> 2,652516957 </td><td> 0,000206279 </td></tr><tr><td> Transkribiert Sequenzen </td><td> BG076361 </td><td> 1460101_at </td><td></td><td> 5.657735 </td><td> 2.5015E-06 </td><td></td><td> 1,296248831 </td><td> 0.22870031 </td></tr><tr><td> spondin 1, (f-spondin) der extrazellulären Matrix Protein </td><td> BC020531 </td><td> 1424415_s_at </td><td></td><td> 0.17783576 </td><td> 1.01658E-06 </td><td></td><td> 0,836181248 </td><td> 0,001380141 </td></tr><tr><td> Calbindin-28K </td><td> BB246032 </td><td> 1448738_at </td><td></td><td> 0,180317904 </td><td> 1.35961E-05 </td><td></td><td> 0,647334052 </td><td> 4.12268E-09 </td></tr><tr><td> MARCKS-like 1 </td><td> AV110584 </td><td> 1437226_x_at </td><td></td><td> 0,186235935 </td><td> 1.47067E-06 </td><td></td><td> 0,499291387 </td><td> 2.34984E-08 </td></tr><tr><td> Matrilin 2 </td><td> BB338441 </td><td> 1455978_a_at </td><td></td><td> 0,187783528 </td><td> 6.19122E-05 </td><td></td><td> 0.8967688 </td><td> 0,282337853 </td></tr><tr><td> Matrilin 2 </td><td> BC005429 </td><td> 1419442_at </td><td></td><td> 0,188195795 </td><td> 0,000105295 </td><td></td><td> 0,915528892 </td><td> 0.35282097 </td></tr><tr><td> spondin 1, (f-spondin) der extrazellulären Matrix Protein </td><td> BQ175871 </td><td> 1442613_at </td><td></td><td> 0,189956563 </td><td> 9.41195E-06 </td><td></td><td> 0,861033222 </td><td> 0.1394266 </td></tr><tr><td> Arrestin 3, retinale </td><td> NM_133205 </td><td> 1450329_a_at </td><td></td><td> 5.2130346 </td><td> 2.90599E-05 </td><td></td><td> 3,944218329 </td><td> 1.07437E-07 </td></tr><tr><td> RIKEN cDNA A330050F15 Gen </td><td> AV325555 </td><td> 1457558_at </td><td></td><td> 0.19186781 </td><td> 0,000119342 </td><td></td><td> 0,660282035 </td><td> 2.47342E-05 </td></tr><tr><td> Ansprechpartnerin 3 </td><td> BB559510 </td><td> 1438628_x_at </td><td></td><td> 0,194404608 </td><td> 4.08641E-07 </td><td></td><td> 0,918742591 </td><td> 0,022545297 </td></tr><tr><td> Calbindin-28K </td><td> BB246032 </td><td> 1417504_at </td><td></td><td> 0,196381321 </td><td> 2.24182E-05 </td><td></td><td> 0,619305124 </td><td> 3.6222E-06 </td></tr><tr><td> Gastrin Releasing Peptide </td><td> BC024515 </td><td> 1424525_at </td><td></td><td> 4.9436426 </td><td> 3.00588E-05 </td><td></td><td> 2,752845903 </td><td> 5.72954E-07 </td></tr><tr><td> Sortilin-bezogene VPS10 domain containing receptor 3 </td><td> AK018111 </td><td> 1425111_at </td><td></td><td> 4.885766 </td><td> 1.03645E-05 </td><td></td><td> 1.29051599 </td><td> 0,029733649 </td></tr><tr><td> Dopamin-Rezeptor-D1A </td><td> BE957273 </td><td> 1455629_at </td><td></td><td> 4.869493 </td><td> 3.77525E-05 </td><td></td><td> 1,815881979 </td><td> 0,000516498 </td></tr><tr><td> Proproteinkonvertase Subtilisin / kexin Typ 5 </td><td> BB241731 </td><td> 1437339_s_at </td><td></td><td> 0,210528027 </td><td> 7.83039E-05 </td><td></td><td> 0,574126078 </td><td> 9.15496E-05 </td></tr></tbody><tbody><tr><td> Interleukin-1 Rezeptor Typ I </td><td> NM_008362 </td><td> 1448950_at </td><td></td><td> 0,210572243 </td><td> 9.64524E-06 </td><td></td><td> 0,241135352 </td><td> 2.79816E-08 </td></tr></tbody></table>

dissection of hippocampal dentate gyrus from adult mouse

Der Hippocampus ist eine der am häufigsten untersuchten Areale im Gehirn wegen der wichtige funktionelle Rolle in Memory-Processing und Lernen, seine bemerkenswerte Nervenzellen Plastizität und ihr Engagement in der Epilepsie, neurodegenerative Erkrankungen und psychische Störungen. Der Hippocampus ist der unterschiedliche Regionen zusammen, Gyrus dentatus, der hauptsächlich aus Nervenzellen Granulat und Ammonshorn, die vor allem Pyramidenneuronen umfasst, und den beiden Regionen werden sowohl anatomische und funktionelle Schaltungen verbunden. Viele verschiedene mRNAs und Proteine ​​werden selektiv im Gyrus dentatus exprimiert, und Gyrus dentatus ist eine Seite der adulten Neurogenese, das heißt, neue Neuronen sind ständig im erwachsenen Gyrus dentatus generiert. Um zu untersuchen, mRNA und Protein-Expression spezifisch für den Gyrus dentatus, ist die Laser-Mikrodissektion häufig verwendet. Diese Methode hat jedoch einige Einschränkungen, wie zum Beispiel die Notwendigkeit für spezielle Apparate und komplizierte Handhabung. In diesem Video aufgezeichnet Protokoll, zeigen wir eine Dissektionstechnik zum Entfernen des Gyrus dentatus aus adulten Maus unter einem Stereomikroskop. Gyrus dentatus Proben unter Verwendung dieser Technik eignen sich für jede Assay, einschließlich Transkriptom-, Proteom-und Zellbiologie Analysen. Wir haben bestätigt, dass die präparierten Gewebe Gyrus dentatus ist durch die Durchführung real-time PCR des Gyrus dentatus-spezifische Gene, Tryptophan 2,3-Dioxygenase (TDO2) und Desmoplakin (DSP) und Ammonshorn bereichert Gene, Meis-related gene 1b (Mrg1b ) und TYRO3 Protein-Tyrosin-Kinase-3 (Tyro3). Die mRNA Expression von TDO2 und DSP im Gyrus dentatus Proben wurden bei offensichtlich höhere erkannt, während Mrg1b und Tyro3 wurden niedrigere, als die im Ammonshorn Proben. Zur Demonstration der Vorteil dieser Methode führten wir DNA-Mikroarray-Analyse unter Verwendung von Proben der gesamten Hippocampus und Gyrus dentatus. Die mRNA-Expression von TDO2 und DSP, die selektiv im Gyrus dentatus exprimiert, in der gesamten Hippocampus von alpha-CaMKII + / - Mäusen zeigte 0,037 und 0,10-fache Veränderungen im Vergleich zu der Wildtyp-Mäusen, bzw.. In der isolierten Gyrus dentatus, jedoch zeigten diese Ausdrücke 0,011 und 0,021-fache Veränderungen im Vergleich zu der Wildtyp-Mäusen, die zeigen, dass Veränderungen der Genexpression im Gyrus dentatus mit größerer Empfindlichkeit nachgewiesen werden. Zusammengenommen können diese bequeme und genaue Dissektionstechnik zuverlässig für Studien über den Gyrus dentatus konzentriert eingesetzt werden.

Watch this Scientific Journal Video about Dissection of Hippocampal Dentate Gyrus from Adult Mouse at JoVE.com

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Ein Dissektionstechnik zur Entfernung des Gyrus dentatus aus adulten Maus unter einem Stereomikroskop wurde in diesem Video aufgezeichnet Protokoll demonstriert.

Dissection der hippocampalen von erwachsenen Maus

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

dissection-of-hippocampal-dentate-gyrus-from-adult-mouse

Research

JoVE Journal

Biology

82.2K Aufrufe.  Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST). Ein Dissektionstechnik zur Entfernung des Gyrus dentatus aus adulten Maus unter einem Stereomikroskop wurde in diesem Video aufgezeichnet Protokoll demonstriert.

Serie: Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissection von 6,5 dpc muriner Embryonen

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Forschung

Biologie

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissection von Organen aus dem Adult Zebrafisch

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Präparation Oenocytes von Adult Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Präparation des Adult Zebrafisch Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Herz Dissection in Larven, juvenile und adulte Zebrafisch, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolierung, Charakterisierung und Differenzierung von kardialen Stammzellen von Erwachsenen Maus Herzen

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolierung und Kultur von primären oralen Keratinozyten aus dem erwachsenen Mausgaumen

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.