Die Autoren bedanken sich bei Dr. Rizaldy Scott am Mount Sinai Hospital in Toronto, Ontario danken für das Geschenk des primären Rac1 flox / flox embryonalen Maus-Fibroblasten. Diese Arbeit wurde durch Betriebskostenzuschüsse an NJ von der Canadian Institutes of Health Research (CIHR, MOP 2009-93526) und dem Natural Science and Engineering Research Council of Canada (NSERC, RG 327372-06) unterstützt. SH und MW werden von CGSMA und CGS-M Auszeichnungen von CIHR und NSERC bzw. unterstützt. Steve Hawley und Melanie Wills trugen gleichermaßen zu dieser Veröffentlichung.

Auftauen von Zellen <ol><li> Erhalten eingefroren embryonalen Maus-Fibroblasten (MEFs) (zuvor aus einzelnen Embryos primären Kulturen generiert) von flüssigem Stickstoff. </li><li> Thaw Zellen durch Eintauchen Fläschchen in 37 ° C Wasser mit wirbelnden, bis Inhalt vollständig aufgetaut. </li><li> Add 9 mL DMEM mit 10% fötalem Rinderserum und 1% Penicillin-Streptomycin zu einer 10cm Zellkulturplatte ergänzt wurde. </li><li> Fügen Sie die aufgetaute Zellsuspension tropfenweise auf die Platte, Rock die Platte vorsichtig, um Zellen und Platzteller in einem 37 ° C, 5% CO 2-Inkubator über Nacht zu zerstreuen. Beachten Sie, dass, wenn die Zellen empfindlich auf DMSO (aus dem Einfrieren Prozess) sind erste Medien mit frischen Medien ersetzt werden kann, nachdem die Zellen an der Platte (ca. 4 Stunden post plating) haften geblieben sind. Beachten Sie auch, dass Zusammenfluss der Zellen nach dem Auftauen ist abhängig von der Anzahl der Zellen eingefroren, die Erholung der Zellen nach dem Einfrieren und die Wachstumsrate der Zellen. </li></ol> Passagierung Zellen <ol start="5"><li> Wenn die Zellen zu praktisch 100% Konfluenz (dh: am nächsten Tag) gewachsen, absaugen den Medien und waschen Sie die Zellen mit 5 ml sterilem PBS. </li><li> Entfernen PBS und 1ml von Trypsin mit 10 mM EDTA und Platzteller in Inkubator für ungefähr 5 Minuten, damit sich Zellen von der Platte zu lösen. </li><li> Wenn die Zellen abgelöst haben, fügen 9 mL der Medien auf die Trypsin-Zellen, Pipette 2-3 mal um Klumpen zu zerstreuen, 1ml dieser Suspension tropfenweise auf eine neue Platte mit 9 mL der Medien, und legen Sie diese in die 37 ° C Inkubator über Nacht . Die Anwesenheit von FBS in den Medien wird das Trypsin alternativ zu inaktivieren, können Zellen zuerst gewaschen werden, um zu verdünnen die Trypsin. </li></ol> Zählen und Beschichtung von Zellen auf Deckgläser <ol start="8"><li> Entfernen Zellen aus Inkubator, waschen und trypsinize wie vorher, nur dieses Mal add 5-6mL Medien zurück zu den trypsiniert Zellen anstelle von 9 mL. </li><li> Pipette Zellen gründlich, um eine vollständige Dissoziation in einer einzigen Zellsuspension zu gewährleisten, und entfernen Sie 15μL mit 15μL von Trypanblau in einem separaten Mikrozentrifugenröhrchen kombinieren. </li><li> Mix dieser Zelle / Trypanblau Suspension durch Auf-und Abpipettieren mehrmals, und fügen Sie 15μL der Zählkammer. </li><li> Unter dem Mikroskop wird abgestorbenen Zellen erscheinen blau. Zählt die Anzahl der klar / farblos (Wohn-) Zellen in einer der 4X4 Gitter auf der Zählkammer. Wiederholen Sie diesen zählen die drei anderen 4X4 Grids (bezeichnet A, B, C und D) und verwenden Sie die folgende Gleichung, um die Anzahl der Zellen pro Mikroliter Suspension zu berechnen: <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="Gleichung 1" /></li><li> Durch Protokoll-Optimierung, haben wir festgestellt, dass plating 30.000 Zellen Ergebnisse in geeigneter Zelldichte für die Visualisierung am Ende des Experiments. </li><li> Zur Berechnung des Volumens der Zellsuspension für 30.000 Zellen benötigt, verwenden Sie die folgende Gleichung: <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="Gleichung 2" /></li><li> Legen Sie nun ein steriles Deckglas in jedes Well einer sechs gut Schüssel und fügen 2 ml Medium in jede Vertiefung, und dann tropfenweise aus Ihrer Zellsuspension das Volumen für 30.000 Zellen benötigt in jedes Well. Beachten Sie, dass Glasdeckgläschen für dieses Experiment verwendet werden, wie wir die Visualisierung werden die Zellen mit Hilfe der Fluoreszenzmikroskopie zu einem späteren Zeitpunkt, da nicht alle Anwendungen werden notwendigerweise Mikroskopie und decken somit rutscht nicht erforderlich sein. </li></ol> Virus Transduction <ol start="15"><li> Nach mehreren Stunden oder über Nacht (genügend Zeit, um sicherzustellen, dass die Zellen auf die Deckgläschen haften), zu entfernen Medien und waschen Sie die Zellen mit 1 ml serumfreiem DMEM. </li><li> 1ml von serum-freien Medien in jede Vertiefung für die Zugabe des Virus. Für dieses Experiment wurde Adeno-Cre-Virus kommerziell von Vector Biolabs erhalten. </li><li> Vorherige Protokoll-Optimierung hat gezeigt, dass eine Vielzahl der Infektion (MOI) von 500 hocheffiziente Gen-Transduktion bietet in primäre MEFs mit wenig oder keinen Einfluss auf die Lebensfähigkeit der Zellen. MOI kann wie folgt berechnet werden: <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="Gleichung 3" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="Gleichung 4" /></li><li> Fügen Sie das berechnete Volumen der Virus direkt in jede Vertiefung, die infiziert ist, schaukeln die Platte, um das Virus und Ort Zellen in die 37 ° C Inkubator über Nacht zu zerstreuen. </li><li> Am nächsten Morgen, entfernen Sie den Virus-haltigen Medien aus den Zellen und waschen Sie die Zellen in 1ml steriles PBS und fügen Sie dann 2 ml regulären Medien in jede Vertiefung. </li><li> Im Falle der Rac1-Zellen in diesem Experiment veranschaulicht, trat morphologischen Veränderungen in den Zellen bei etwa 72 Stunden nach der Transduktion. </li></ol> Repräsentative Ergebnisse Die Zellen wurden visualisierend durch Färbung mit dem Aktin-Farbstoff Phalloidin (für die Bildgebung dient nur) und abgebildet mit dem Leica TCS SP5-Multiphotonen konfokalen Laser-Scanning-Mikroskop an der Advanced Analysis Centre an der University of Guelph. Abbildung 1 zeigt die kontrahierte und längliche Morphologie der Rac1 flox / flox Zellen, Adeno-Cre-Virus im Vergleich zu den gleichen Zellen nicht zu Virus ausgesetzt wurden ausgesetzt. <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Vergleich zwischen Rac1 flox / flox Zellen mit Adeno-Cre-Virus (links) im Vergleich zu nicht infizierten Zellen aus der gleichen Quelle (rechts) infiziert.

<ol>
	<li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li><li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. o. w. n. e. y., Dinauer, M., Kwiatkowski, 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=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li><li>Prost, S., Sheahan, S., Rannie, D., Harrison, 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=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li><li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li><li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, 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=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li><li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li></ol>

Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her durch die Canadian Council on Animal Care Set durchgeführt.

Das dazugehörige Video zeigt, wie ein rekombinantes Virus verbrauchsteuerpflichtige ein bestimmtes Gen in vitro mit Cre-Rekombinase verwendet werden kann. Die Entwicklung dieses Protokoll jedoch hat offenbaren einige spezielle Punkte wert Adressierung. <ul><li> Proper Sicherheitsvorkehrungen sollten immer dann eingesetzt, wenn mit Adenoviren werden. Ein Laborkittel und Handschuhe sollten immer getragen werden, während der Arbeit mit dem Virus. Medien und Plastmetall sollte mit 10% Bleichmittel für mindestens 30 Minuten behandelt werden, und Plastmetall sollte danach autoklaviert werden. </li><li> Vermeiden Sie wiederholtes Einfrieren und Auftauen des Virus Lager, da dies Virustiter beeinflussen und reduzieren somit Transduktionseffizienz. Kleine Mengen des Virus Lager sollten zunächst in separaten Röhren aliquotiert werden. </li><li> MOIs reichen von 5 bis 50 wurden während der Methodenentwicklung getestet und effiziente Transduktion Raten wurden in den meisten Fällen beobachtet, ohne negative Auswirkungen auf die Lebensfähigkeit der Zellen. </li><li> Adeno-GFP wurde auch während der Methodenentwicklung zur schnellen Bildschirm Transduktion Effizienz. Diese Steuerung (oder einen leeren Vektor) sollte während des Experiments, dass virale Infektion allein sicher durchgeführt werden nicht in zellulären Veränderungen führen. Auch wenn Auslösen der floxed Gen von Interesse in Ihren Zellen in keiner zellulären Veränderungen (dh Veränderungen in Morphologie oder Lebensfähigkeit) Ergebnis, dann infizieren den gleichen Zelltyp mit Adeno-GFP ist ein wichtiges Steuerelement zu verwenden, um nachzuweisen, dass Viren Transduktion ist auftreten. </li></ul> Über die Untersuchung von Signalkaskaden in Kultur, hat das Adeno-Cre-Ansatz auch in vivo eingesetzt worden, um unter bestimmten Bedingungen zu entfernen Gene von Versuchstieren 3,4 und auf bestimmte Populationen von Zellen innerhalb eines Organismus 6 Beschriften. Das Adenovirus-System kann auch angepasst an anderer Gene als Cre-Rekombinase in Wirtszellen einzuführen. Zwei wesentliche Vorteile bei der Verwendung dieser Lieferung Vektors sind seine hohe Transduktion und Gentransfer Effizienz und mangelnde Integration mit Wirts-DNA. Allerdings kann die Generierung von neuen rekombinanten Adenoviren werden arbeitsintensiv. Die hier beschriebene Protokoll daher nutzt die Kraft des adenoviralen Systems durch die Nutzung der bisher entwickelten Adeno-Cre-Virus und Kupplung, die mit unseren floxed Allel von Interesse, Rac1. Durch diese bedingte knock out Experiment haben wir bestätigt, dass Transduktion mit dem Adeno-Cre Virus MEFs Tragen einer floxed Rac1-Allels in der Tat dazu führen Exzision Rac1 führt zu Veränderungen in der Zellmorphologie charakteristisch für Rac1-defizienten Zellen 2,5.

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<td>Ad-CMV-Cre virus</td>
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adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

Die Fähigkeit, genetisch bestimmte Komponenten entfernen von verschiedenen Zell-Signalkaskaden verfügt über ein integriertes Werkzeug in modernen Signaltransduktion Analyse. Eine besondere Methode, um diese bedingte Löschung zu erreichen, ist über die Verwendung des Cre-loxP-System. Dieses Verfahren beinhaltet flankierende das Gen von Interesse mit loxP-Stellen, die spezifischen Erkennungssequenzen für die Cre-Rekombinase-Protein. Die Exposition der sogenannten floxed (flankiert von loxP site) DNA, dieses Enzym zu einer Cre-vermittelte Rekombination Veranstaltung in der loxP-Stellen und die anschließende Entfernung der dazwischenliegenden Gen 3. Mehrere verschiedene Methoden existieren, um die Rekombinase Cre auf der Website von Interesse zu verwalten. In diesem Video zeigen wir die Verwendung eines Adenovirus mit der Cre-Rekombinase-Gen zur primären embryonalen Maus-Fibroblasten (MEFs) von Embryonen mit einem floxed Rac1 Allel 1 erhalten zu infizieren. Unsere Gründe für die Auswahl Rac1 MEFs für unsere Experimente, dass eine klare morphologische Veränderungen können auf Streichung von Rac1, aufgrund von Änderungen in der Aktin-Zytoskelett 2,5 gesehen werden. 72 Stunden nach der viralen Transduktion und Cre Expression wurden die Zellen gefärbt mit dem Aktin-Farbstoff Phalloidin und abgebildet mit Hilfe der konfokalen Laser Scanning Mikroskopie. Es wurde beobachtet, dass MEFs, die dem Adeno-Cre-Virus ausgesetzt waren erschienen unter Vertrag und verlängerte in der Morphologie im Vergleich zu nicht infizierten Zellen, im Einklang mit früheren Berichten 2,5. Das Adenovirus Methode der Rekombinase Cre Lieferung ist vorteilhaft, da die Adeno-Cre-Virus ist leicht zugänglich und Gendeletion via Cre in nahezu 100% der Zellen kann mit optimierter adenoviralen Infektion erzielt werden.

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

In diesem Video verwenden wir ein Adenovirus Durchführung der Cre-Rekombinase-Gen zur primären embryonalen Maus-Fibroblasten Tragen einer floxed Rac1 Allel zu infizieren.

Adenovirus-vermittelte genetische Entfernung von Signalmolekülen in kultivierten primären embryonalen Maus-Fibroblasten

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

Research

JoVE Journal

Biology

25.0K Aufrufe.  University of Guelph. In diesem Video verwenden wir ein Adenovirus Durchführung der Cre-Rekombinase-Gen zur primären embryonalen Maus-Fibroblasten Tragen einer floxed Rac1 Allel zu infizieren.

Serie: Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Live-Imaging von Dense-Core-Vesikel in Primary hippokampalen Neuronen

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Forschung

Biologie

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung embryonalen Maus-Fibroblasten mit einem Vier Transcription Factor, induzierbare Doxycycline Lentivirale Transduktionssystems

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

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 remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

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 ...

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Testen der Fähigkeit des diffusiblen Signalmoleküle zu embryonalen Spinal kommissuralen Axone neu ausrichten

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Schnelle Fibroblast Entfernung aus High Density humanen embryonalen Stammzellen Kulturen

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Schnelle Genetische Analyse von epithelial-mesenchymale Signaling Während Haarregeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Organell-Transport in kultivierten<em&gt; Drosophila</em&gt; Zellen: S2-Zelllinie und primären Neuronen.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Glukoseaufnahme Messung und Response auf Insulin Stimulation in<em&gt; In vitro</em&gt; Kultivierte menschliche primäre Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-innere Zelle Masse Injektion von embryonalen Stammzellen führt zu höheren Chimerism Raten

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Adenovirus-vermittelte genetische Entfernung von Signalmolekülen in kultivierten primären embryonalen Maus-Fibroblasten

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