Gli autori desiderano ringraziare il Dr. Scott Rizaldy al Mount Sinai Hospital di Toronto per il dono del primario Rac1 flox / flox topo fibroblasti embrionali. Questo lavoro è stato supportato da sovvenzioni di funzionamento a New Jersey dal Canadian Institutes of Health Research (CIHR, MOP 2009-93526) e Scienze Naturali e Ingegneria Research Council del Canada (NSERC, RG 327372-06). SH e MW sono supportati da CGSMA e CGS-M premi da CIHR e NSERC, rispettivamente. Steve Wills Hawley e Melanie contribuito in maniera uguale a questo articolo.

Celle di scongelamento <ol><li> Ottenere fibroblasti di topo embrioni congelati (MEF) (generato in precedenza da colture di singolo embrione primaria) da azoto liquido. </li><li> Scongelare le cellule immergendo flaconcino in acqua a 37 ° C, rimestando fino a quando i contenuti sono completamente scongelati. </li><li> Aggiungere 9 ml di DMEM che è stato integrato con il 10% di siero fetale bovino e 1% di penicillina-streptomicina ad una piastra di coltura cellulare 10 centimetri. </li><li> Aggiungere la sospensione cellulare scongelati goccia a goccia alla piastra, il rock delicatamente la piastra per disperdere le cellule e conservare in un CO 37 ° C, 5% 2 incubatore notte. Si noti che se le cellule sono sensibili a DMSO (dal processo di congelamento), i media iniziale può essere sostituita con mezzi freschi dopo che le cellule hanno aderito alla piastra (circa quattro ore dopo la placcatura). Si noti inoltre che la confluenza delle cellule dopo lo scongelamento dipende dal numero di cellule congelate, il tasso di recupero delle cellule dopo il congelamento, e il tasso di crescita delle cellule. </li></ol> Cellule Passaging <ol start="5"><li> Quando le cellule sono cresciuti essenzialmente al 100% di confluenza (cioè il giorno successivo), aspirare al largo della media e lavare le cellule con 5 ml di PBS sterile. </li><li> Rimuovere PBS e aggiungere 1 ml di tripsina contenente 10mM EDTA e conservare in incubatrice per circa 5 minuti per consentire alle cellule di staccarsi dalla piastra. </li><li> Quando le cellule sono staccati, aggiungere 9 ml di supporto alle cellule trypsinized, pipetta 2-3 volte per disperdere i grumi, aggiungere 1 ml di questa sospensione goccia a goccia su un piatto nuovo che contiene 9ml di media, e questo luogo nel 37 ° C durante la notte incubatore . La presenza di FBS nei media si inattivare la tripsina, in alternativa, le cellule possono essere lavati prima di diluire la tripsina. </li></ol> Conteggio e placcatura cellule su vetrini coprioggetto <ol start="8"><li> Rimuovere le cellule da incubatore, lavare e trypsinize come prima, ma questa volta aggiungere 5-6 ml mezzi di comunicazione di nuovo alle cellule trypsinized invece di 9 ml. </li><li> Cellule pipetta a fondo per garantire la completa dissociazione in una sospensione singola cella, e rimuovere 15μL da abbinare a 15μL di tripano blu in una provetta per microcentrifuga separata. </li><li> Mescolare questa cella / trypan sospensione blu pipettando su e giù parecchie volte, e aggiungere 15μL al emocitometro. </li><li> Al microscopio, le cellule morte apparirà blu. Contare il numero di cellule chiare / incolore (vivente) in una delle griglie 4x4 sul emocitometro. Ripetere questa contare su tre griglie altri 4X4 (indicati A, B, C e D) e utilizzare la seguente equazione per calcolare il numero di cellule per ml di sospensione: <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="Equazione 1" /></li><li> Attraverso l&#39;ottimizzazione del protocollo, abbiamo stabilito che placcatura 30.000 risultati cellule della densità cella appropriata per la visualizzazione a conclusione dell&#39;esperimento. </li><li> Per calcolare il volume di sospensione cellulare necessaria per 30.000 celle, utilizzare la seguente equazione: <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="Equazione 2" /></li><li> Ora, mettere un coperchio di vetro sterile scivolare in ciascun pozzetto di un piatto ben sei e aggiungere 2 ml di media per ogni bene, e quindi aggiungere goccia a goccia dalla sospensione cellulare il volume necessario per 30.000 cellule in ciascun pozzetto. Si noti che scivola copertura in vetro vengono utilizzati per questo esperimento come saremo visualizzare le cellule al microscopio a fluorescenza in un punto secondo momento, non tutte le applicazioni saranno necessariamente microscopia, e quindi coprire scivola potrebbe non essere necessario. </li></ol> Trasduzione del virus <ol start="15"><li> Dopo diverse ore o durante la notte (tempo utile per assicurare che le cellule aderiscono al scivola copertina), rimuovere i supporti e lavare le cellule con 1 ml di DMEM senza siero. </li><li> Aggiungere 1 ml di siero-media liberi in ciascun pozzetto per l&#39;aggiunta del virus. Per questo esperimento, Cre adeno-virus è stato ottenuto commercialmente da Biolabs Vector. </li><li> Ottimizzazione del protocollo precedente ha dimostrato che una molteplicità di infezione (MOI) di 500 offre trasduzione del gene altamente efficiente in primari MEF con poco o nessun effetto sulla vitalità cellulare. MOI può essere calcolato come segue: <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="Equazione 3" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="Equazione 4" /></li><li> Aggiungere il volume calcolato di virus direttamente a ogni bene che deve essere infetto, agitare delicatamente la piastra per disperdere le cellule virus e posto in incubatore a 37 ° C durante la notte. </li><li> La mattina successiva, rimuovere il virus contenente i media dalle cellule e lavare le cellule in 1 ml di PBS sterile e poi aggiungere 2 ml di mezzi regolari per ciascun pozzetto. </li><li> Nel caso delle cellule Rac1 esemplificato in questo esperimento, cambiamenti morfologici avvenuti nelle cellule in circa 72 ore di trasduzione del messaggio. </li></ol> Rappresentante Risultati Le cellule sono state visualizzated dalla colorazione con il colorante actina falloidina (per scopi di imaging solo) e ripreso con il TCS-SP5 Leica multiphoton microscopio confocale a scansione laser presso il Centro di analisi avanzata presso l&#39;Università di Guelph. La figura 1 mostra la morfologia contratta e allungata del Rac1 flox / flox cellule che sono state esposte a virus adeno-Cre rispetto alle stesse cellule non esposte al virus. <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="Figura 1" /> Figura 1. Confronto tra Rac1 flox / flox cellule infettate con virus adeno-Cre (a sinistra) rispetto a cellule non infettate dalla stessa fonte (a destra).

<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>

Gli esperimenti sugli animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti dal Consiglio canadese di prodotti per animali.

Il video che accompagna un esempio di come un virus ricombinante può essere usato per accise uno specifico gene in vitro utilizzando Cre ricombinasi. Lo sviluppo di questo protocollo tuttavia ha rivelato alcuni particolari punti vale la pena affrontare. <ul><li> Procedure di sicurezza devono sempre essere impiegato quando si lavora con gli adenovirus. Un camice da laboratorio e guanti devono essere indossati in ogni momento mentre si lavora con il virus. Media e articoli in plastica devono essere trattati con il 10% di candeggina per almeno 30 minuti, e articoli in plastica devono essere sterilizzati in autoclave successivamente. </li><li> Evitare ripetuti di congelamento-scongelamento dello stock del virus come questo può influenzare il titolo del virus e quindi ridurre l&#39;efficienza di trasduzione. Piccoli volumi di magazzino virus dovrebbe inizialmente essere aliquotati in tubi separati. </li><li> MOIS che vanno dal 5-50 sono state testate durante lo sviluppo del metodo e dei tassi di trasduzione efficiente sono stati osservati nella maggior parte dei casi, senza effetti negativi sulla vitalità cellulare. </li><li> Adeno-GFP è stata utilizzata anche durante lo sviluppo del metodo per lo screening rapido l&#39;efficienza di trasduzione. Questo controllo (o un vettore vuoto) deve essere effettuato durante l&#39;esperimento per garantire che l&#39;infezione virale da solo non porta a cambiamenti cellulari. Allo stesso modo, se floxed ablazione del gene di interesse nelle vostre cellule non comportare alcun cambiamento cellulare (ad esempio i cambiamenti nella morfologia e vitalità), quindi infettare lo stesso tipo di cella con adeno-GFP è un controllo importante da utilizzare per dimostrare che la trasduzione del virus è che si verificano. </li></ul> Al di là l&#39;esame della segnalazione cascate nella cultura, l&#39;adeno-Cre approccio è stato utilizzato in vivo per rimuovere condizionalmente geni di animali da esperimento 3,4, e di etichettare specifiche popolazioni di cellule all&#39;interno di un organismo 6. Il sistema di adenovirus può essere adattato anche per introdurre geni diversi da Cre ricombinasi nelle cellule ospiti. Due principali vantaggi dell&#39;utilizzo di questo vettore di consegna sono di trasduzione del suo alto rendimento e la consegna del gene, e la mancanza di integrazione con il DNA ospite. Tuttavia, la generazione di nuovi adenovirus ricombinante può essere laborioso. Il protocollo qui descritto sfrutta quindi la potenza del sistema adenovirale sfruttando l&#39;precedentemente sviluppato Cre adeno-virus, e giunto che con il nostro allele floxed di interesse, Rac1. Attraverso questo bussare condizionale fuori esperimento, abbiamo confermato che trasduzione con il virus adeno-Cre di MEF portando un allele floxed Rac1 effettivamente causa asportazione di Rac1 portare ai cambiamenti nella caratteristica morfologia cellulare di Rac1-deficienti cellule 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

La possibilità di rimuovere geneticamente componenti specifici delle varie cascate segnalazione cellulare è stato uno strumento integrato per l&#39;analisi moderna di trasduzione del segnale. Un particolare metodo per raggiungere questo obiettivo la cancellazione condizionale è attraverso l&#39;uso del Cre-loxP sistema. Questo metodo comporta che fiancheggiano il gene di interesse con i siti loxP, che sono sequenze di riconoscimento specifico per la proteina ricombinasi Cre. Esposizione del cosiddetto floxed (affiancato da sito loxP) DNA a questo risultato in un enzima Cre-mediata evento di ricombinazione nei siti loxP, e conseguente asportazione del gene intervenire 3. Esistono diversi metodi per amministrare Cre ricombinasi al sito di interesse. In questo video, che illustrano l&#39;utilizzo di un adenovirus contenente il gene Cre ricombinasi per infettare i fibroblasti embrionali del mouse primario (MEF) ottenute da embrioni contenenti un floxed Rac1 allele 1. La nostra logica per la selezione Rac1 MEF per i nostri esperimenti è chiaro che i cambiamenti morfologici possono essere visti su cancellazione di Rac1, a causa di alterazioni del citoscheletro di actina 2,5. 72 ore dopo la trasduzione virale ed espressione Cre, le cellule sono state colorate con il colorante falloidina actina e ripreso utilizzando la microscopia confocale a scansione laser. È stato osservato che MEF che erano stati esposti al virus adeno-Cre apparsa contratta e allungata nella morfologia rispetto alle cellule non infette, in coerenza con le relazioni precedenti 2,5. Il metodo di consegna adenovirus Cre ricombinasi è vantaggioso come il Cre adeno-virus è facilmente disponibile, e l&#39;eliminazione del gene via Cre in quasi il 100% delle cellule può essere realizzato con ottimizzati infezione adenovirale.

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 questo video possiamo utilizzare un adenovirus che trasportano il gene Cre ricombinasi per infettare i fibroblasti di topo primario embrionale porta un allele floxed Rac1.

Adenovirus-mediata rimozione genetica di molecole di segnalazione in fibroblasti di topo coltivate primarie embrionali

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

24.8K Views.  University of Guelph. In questo video possiamo utilizzare un adenovirus che trasportano il gene Cre ricombinasi per infettare i fibroblasti di topo primario embrionale porta un allele floxed Rac1.

Video: 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.

Immagini dal vivo di Dense-core Vescicole nella Primaria neuroni coltivati ​​ippocampale

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

Ricerca

Biologia

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.

Generazione di cellule staminali pluripotenti indotte da Riprogrammazione fibroblasti embrionali di topo con un fattore quattro trascrizione, Doxiciclina inducibile lentivirali sistema di trasduzione

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

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.

Capacità di analizzare il diffusibili molecole di segnalazione per l&#39;orientamento embrionali assoni spinali commissurali

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.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

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.

Fibroblast rimozione veloce da alta densità Culture staminali embrionali umane

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

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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.

Rapida analisi genetica di epitelio-mesenchimale segnalazione durante la rigenerazione dei capelli

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.

Organelle Trasporti in Colta<em&gt; Drosophila</em&gt; Celle: Cell Line S2 e Primaria neuroni.

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.

Misurazione e risposta alla somministrazione di glucosio alla stimolazione dell&#39;insulina in<em&gt; In vitro</em&gt; Myotubes primario umano coltivato

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-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

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