Desideriamo ringraziare Drs. Meera Saxena e Jonathan Mathias per gli utili commenti sul manoscritto. Ringraziamo Drs. Michael Parsons, Pamela Raymond, e Rachel Wong per la fornitura di linee transgeniche. Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dal protocollo approvato IACUC MCG 07-12-003 * B. Questo lavoro è stato sostenuto da NIH R21 MH083614 e March of Dimes Basilio Scholar Award O Conner Starter per JSM

<ol>
	<li>K&#246;ster, R. W., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+microscopy+of+brain+development.">Time-lapse microscopy of brain development.</a> Methods Cell Biol. 76, 207-235 (2004).</li><li>Mumm, J. S., Williams, P. R., Godinho, L., Koerber, A., Pittman, A. J., Roeser, T., Chien, C. B., Baier, H., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+dendritic+targeting+of+laminated+afferents+by+zebrafish+retinal+ganglion+cells.">In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells.</a> Neuron. 52, 609-621 (2006).</li><li>Curado, S., Anderson, R. M., Jungblut, B., Mumm, J., Schroeter, E., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> Dev Dyn. 236, 1025-1035 (2007).</li><li>Ren, J. Q., McCarthy, W. R., Zhang, H., Adolph, A. R., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+visual+responses+of+wild-type+and+hypopigmented+zebrafish.">Behavioral visual responses of wild-type and hypopigmented zebrafish.</a> Vision Res. 42, 293-299 (2002).</li><li>Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., Parsons, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+ablation+of+beta+cells+in+the+embryonic+zebrafish+pancreas+using+E.+Coli+nitroreductase.">Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. Coli nitroreductase.</a> Mech Dev. 124, 218-229 (2007).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Vis Exp. , (2010).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Vis Exp. , (2009).</li></ol>

JSM ha ottenuto un brevetto sull&#39;utilizzo della nitroreductase sistema basato su di ablazione delle cellule in zebrafish (Mumm JS, e Schroeter, EH ablazione ablazione mirata e regionali cellulare in Zebrafish &quot;US Patent 7514595. 7 aprile 2009). Questa tecnica è utilizzato come piattaforma generale per una serie di strumenti e della biotecnologia società di servizi, Luminomics, Inc., che JSM fondato e rimane una delle parti interessate in

In questo video, una panoramica delle tecniche che usiamo per multicolore confocale time-lapse imaging e l&#39;ablazione delle cellule bersaglio è fornito. Ci avvaliamo di time-lapse imaging per monitorare il comportamento dei diversi tipi di cellule di interesse in vivo, mentre l&#39;ablazione delle cellule bersaglio viene utilizzato per studiare la funzione del circuito e delle cellule neuronali specifici paradigmi rigenerazione neuronale. Gli esempi riportati evidenziano diversi vantaggi che possono essere raccolte da questi approcci. Forse più significativa, time-lapse imaging fornisce un paradigma internamente controllata; fenomeni qualsiasi osservati possono essere legati a stati di nuovo precedenti. Questo permette un confronto diretto, piuttosto che dedurre da effettuare attraverso punti di tempo sperimentali, apportando modifiche relativamente facile da quantificare e riducendo sperimentale &#39;rumore&#39; associata a variazioni all&#39;interno di una popolazione. Un vantaggio di immagine multicolore è la capacità di visualizzare le modifiche ad interagire e / o tipi di cellule vicine. Ad esempio, utilizzando una linea transgenica che le etichette glia Müller, siamo ora che caratterizzano la risposta di questi potenziali lesioni indotte da cellule staminali per la perdita di discreto sottopopolazioni di cellule della retina. Abbiamo anche usato questo metodo per definire nuovi meccanismi di formazione del circuito neurale 2. Infine, oltre agli studi di rigenerazione cellulare, abbiamo recentemente iniziato a utilizzare il NTR sistema basato ablazione per indagare il ruolo funzionale dei sottotipi neuronali così i circuiti neurali separati utilizzando diversi paradigmi comportamentali e analisi elettrofisiologiche. Il vantaggio unico di impiegare il pesce zebra per tali studi è che, grazie alla loro capacità rigenerativa, i deficit indotti sono temporanee. Così, da correlare con time-lapse analisi di immagini si possono caratterizzare i meccanismi che portano alla reinnervazione, così come il grado di reinnervazione necessari per il recupero funzionale. Le modalità previste può essere adattato a molte domande differenti. Per esempio, analisi simili di ablazione e di rigenerazione può essere applicato a qualsiasi sottotipo transgenically definibile cellulare. Collettivamente, questi studi hanno un potenziale di espandere la nostra comprensione della rigenerazione a livello di tipi di cellule individuali ed entro nicchie discrete cellule staminali.

1. Reagenti: Soluzione Danieau di: <ul><li> cloruro di sodio (Fisher, S271 1) </li><li> cloruro di potassio (Fisher, P217 500) </li><li> HEPES (Fisher, BP310500) </li><li> solfato di magnesio (Fisher, M65 500) </li><li> nitrato di calcio (Fisher, C109500) </li><li> Penicillina-streptomicina (Fisher, ICN1670049) </li></ul> Altri reagenti: <ul><li> Bassa temperatura di fusione agarosio (Fisher, BP165-25) </li><li> Tricane metanosolfonato (Fisher, NC9435281) </li><li> N-phenylthiourea (Fisher, NC9968868) </li><li> Metronidazolo (Fisher, AC21034-1000) </li></ul> 2. Forniture: <ul><li> Profonda petri-piatti (Fisher Scientific / Corning, 08-772-32) </li><li> Dumont # 5 pinza sottile (Fisher, NC9404145) </li></ul> 3. Attrezzatura: <ul><li> Zoom stereoscopio (Olympus, SZ51) </li><li> Fluorescenza stereoscopio Zoom (Olympus, SZX16) </li><li> Microscopio confocale (Olympus FV1000) </li><li> Digital riscaldamento blocco - Isotemp (Fisher, 11-715-125DQ) </li><li> Barnstead / Thermolyne Incubatore da banco (Fisher, 11-702-4). </li></ul>

multicolor time-lapse imaging of transgenic zebrafish: visualizing retinal stem cells activated by targeted neuronal cell ablation

Ad alta risoluzione time-lapse imaging di larve di zebrafish vivente può essere utilizzato per visualizzare come i processi biologici si svolgono (per una rassegna vedi 1). Pesce composto transgenici che esprimono diversi reporter fluorescente in tipi di cellule vicine fornire un mezzo di seguire interazioni cellulari 2 e / o tessuto risposte a livello di manipolazioni sperimentali nel corso del tempo. In questo video, dimostriamo metodi che possono essere utilizzati per l&#39;imaging più tipi di cellule transgenically etichettati serialmente nel pesce singoli corsi nel tempo che può estendersi da pochi minuti a diversi giorni. Le tecniche descritte sono applicabili a qualsiasi studio cercando di correlare il &quot;comportamento&quot; della vicina tipi di cellule nel tempo, tra cui: &#39;catch and release&#39; 1) seriale metodi per l&#39;imaging di un gran numero di pesci più giorni consecutivi, 2) metodi semplificati per la separazione fluorofori con sovrapposizione di eccitazione / emissione di profili (ad esempio, GFP e YFP), 3) uso di ipopigmentate linee mutanti di estendere la finestra temporale a disposizione per imaging ad alta risoluzione in ritardo stadi larvali di sviluppo, 4) uso di membrane mirato reporter fluorescente per rivelare multa dettaglio morfologico delle singole cellule così come i dettagli cellulari in grandi popolazioni di cellule, e 5) un metodo precedentemente descritto per chimicamente indotte ablazione di tipi di cellule transgenically mirata, cioè, nitroreductase (NTR) conversione mediata di substrati profarmaco, come il metronidazolo (MTZ), ai derivati ​​citotossici 3,5. Come esempio di questi approcci, si visualizzerà l&#39;ablazione e la rigenerazione di un sottotipo di retina bipolare neurone all&#39;interno di singoli pesci più giorni. Contemporaneamente ci sarà monitorare diversi altri tipi di cellule della retina, compreso vicini e non mirato le cellule bipolari e il potenziale degenerazione stimolate le cellule staminali della retina (ad esempio, glia Mϋller). Questa strategia viene applicata nel nostro laboratorio per la caratterizzazione delle cellule e dei tessuti a livello (ad esempio, di nicchia delle cellule staminali) risposte alla perdita selettiva e la rigenerazione di mirati tipi di cellule neuronali.

Watch this Scientific Journal Video about Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation at JoVE.com

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

In questo video, le tecniche per multicolor confocale time-lapse imaging e ablazione delle cellule bersaglio sono forniti. Time-lapse imaging è utilizzato per monitorare il comportamento dei diversi tipi di cellule di interesse<em&gt; In vivo</em&gt;. L&#39;ablazione delle cellule bersaglio facilita il circuito neurale funzione di studio e cellule specifici paradigmi rigenerazione neuronale.

Multicolor time-lapse imaging di transgenici Zebrafish: Visualizzare cellule staminali della retina Attivato da mirata ablazione delle cellule neuronali

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

multicolor-time-lapse-imaging-transgenic-zebrafish-visualizing

Research

JoVE Journal

Biology

16.3K Views.  Medical College of Georgia. In questo video, le tecniche per multicolor confocale time-lapse imaging e ablazione delle cellule bersaglio sono forniti. Time-lapse imaging è utilizzato per monitorare il comportamento dei diversi tipi di cellule di interesse In vivo. L'ablazione delle cellule bersaglio facilita il circuito neurale funzione di studio e cellule specifici paradigmi rigenerazione neuronale.

Video: Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivazione di cellule staminali embrionali umane da Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Ricerca

Biologia

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Due fotoni assotomia e time-lapse confocale in embrioni di zebrafish vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generazione di Human CD40 delle cellule B attivate

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tessuto Chimere mirate embrionali: Zebrafish gastrula trapianto di cellule

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Mappatura destino di cellule staminali embrionali umane per la formazione teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualizzazione di sviluppo craniofacciale nei SOX10: Kaede transgenici Zebrafish line utilizzando Time-lapse Microscopia confocale

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

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

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

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

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

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

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

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

Generazione di transgenici<em&gt; Hydra</em&gt; Da Embryo microiniezione

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...