Gli autori desiderano ringraziare per l&#39;assistenza tecnica di C. Zambataro durante le riprese di questo protocollo. Noi riconosciamo con gratitudine il sostegno della Fondazione Glenn for Medical Research (SM), La fondazione Hillblom, e il National Institutes of Health per un premio Centro Shock Nathan (P30AG025708) e PO1AG025901. JMF è stato sostenuto da T32AG000266 assegnato al Buck Institute for Research on Aging.

<ol>
	<li>Wolska, B. M., Solaro, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+adult+mouse+cardiac+myocytes+for+studies+of+contraction+and+microfluorimetry.">Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry.</a> Am. J. Physiol. 271 (3 Pt 2), H1250-H1255 (1996).</li><li>Shioya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+isolating+healthy+heart+cells+from+mouse+models.">A simple technique for isolating healthy heart cells from mouse models.</a> J. Physiol. Sci. 57 (6), 327-335 (2007).</li><li>Guo, G., Huss, M., Tong, G. Q., Wang, C., Li Sun, L., Clarke, N. D., Robson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+cell+fate+decisions+revealed+by+single-cell+gene+expression+analysis+from+zygote+to+blastocyst.">Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.</a> Dev. Cell. 18 (4), 675-685 (2010).</li><li>Narsinh, K. H., Sun, N., Sanchez-Freire, V., Lee, A. S., Almeida, P., Hu, S., Jan, T., Wilson, K. D., Leong, D., Rosenberg, J., Yao, M., Robbins, R. C., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> J. Clin. Invest. 121 (3), 1217-1221 (2011).</li><li>D'haene, B., Vandesompele, J., Hellemans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+and+objective+copy+number+profiling+using+real-time+quantitative+PCR.">Accurate and objective copy number profiling using real-time quantitative PCR.</a> Methods. 50 (4), 262-270 (2010).</li><li>Cook, 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=The+Anatomy+of+the+Laboratory+Mouse.">The Anatomy of the Laboratory Mouse.</a> Informatics. , (1965).</li><li>Gaillard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aorta.">Aorta.</a> Radiopaedia. , (2008).</li><li>St&#229;hlberg, A., Andersson, D., Aurelius, J., Faiz, M., Pekna, M., Kubista, M., Pekny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+cell+populations+with+single-cell+gene+expression+profiling:+correlations+and+identification+of+astrocyte+subpopulations.">Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations.</a> Nucleic Acids Res. 39 (4), e24-e24 (2011).</li><li>Zhong, J. F., Chen, Y., Marcus, J. S., Scherer, A., Quake, S. R., Taylor, C. R., Weiner, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+processor+for+gene+expression+profiling+of+single+human+embryonic+stem+cells.">A microfluidic processor for gene expression profiling of single human embryonic stem cells.</a> Lab Chip. 8 (1), 68-74 (2008).</li><li>Bahar, R., Hartmann, C. H., Rodriguez, K. A., Denny, A. D., Busuttil, R. A., Dollé, M. E., Calder, R. B., Chisholm, G. B., Pollock, B. H., Klein, C. A., Vijg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell-to-cell+variation+in+gene+expression+in+ageing+mouse+heart.">Increased cell-to-cell variation in gene expression in ageing mouse heart.</a> Nature. 441 (7096), 1011-1014 (2006).</li><li>Raj, A., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature,+nurture,+or+chance:+stochastic+gene+expression+and+its+consequences.">Nature, nurture, or chance: stochastic gene expression and its consequences.</a> Cell. 135 (2), 216-226 (2008).</li><li>Elowitz, M., Levine, A., Siggia, E., Swain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li></ol>

<p class="jove_content"&gt; Accesso libero e la produzione di questo di questo articolo è sponsorizzato Sigma-Aldrich.</p

Questo metodo ha la possibilità di generare cardiomiociti per una serie di funzionali e studi di espressione genica. L&#39;esame di singole cellule è una zona fiorente in analisi di espressione genica. Il vantaggio di esaminare i livelli di trascrizione a livello della singola cellula è che permette l&#39;esame di una popolazione di cellule pure, che non è possibile dal preparazioni tessuto intero. Inoltre, l&#39;analisi singola cella consente per l&#39;esame di variazione stocastica dei livelli di mRNA nelle cellule individuali per definire popolazioni di cellule che in precedenza erano ritenuti omogenei 8,9. Oltre a identificare i geni che sono potenzialmente stocastici nella loro espressione genica 10,11,12, il metodo consente anche per l&#39;identificazione delle popolazioni cellulari rari definito dai profili gene loro espressione. Mentre questo metodo fornisce una serie di entusiasmanti possibilità di utilizzo in analisi di espressione genica, ci sono somavvertimenti e considerazioni e nell&#39;uso di singole cellule. Il limite principale ad analisi singola cella, è la raccolta di cellule sufficiente per l&#39;esperimento di raggiungere una significatività statistica per quanto riguarda la metrica di interesse, per esempio varianza. Nei casi in cui il numero di cellule isolate dal tessuto di interessi non è una limitazione, si può esaminare centinaia di cellule per gruppo, utilizzando approcci come sequenziamento di prossima generazione, o array nanofluidic. Tuttavia, in alcuni casi, ci possono essere difficoltà a ottenere cellule sufficienti dal tessuto di interesse. Questo può in parte essere causato da procedure momento idiosincratico intensivo per la raccolta del tipo di cellula bersaglio. Tuttavia, un&#39;attenta pianificazione e la preparazione prima di procedere con la raccolta singola cellula può ancora consentire una rigorosa analisi singola cellula con limitato errore tecnico. Quando l&#39;esame dei risultati deve essere considerato, che anche se questa procedura per le celle di isolamento è stato utilizzato in numerose Studies (tra cui microscopia, elettrofisiologia, ecc), lo stesso isolamento potrebbe potenzialmente effetto di espressione genica. Come per qualsiasi metodo che manipola i campioni biologici, i risultati delle indagini svolte deve essere attentamente convalidato per garantire l&#39;espressione singola cella è rappresentativo del tessuto stesso e pregiudizi non tecnico. Metodi come ibridazione in situ può risultare utile per verificare questi risultati nel tessuto intatto. Infine, è fondamentale per garantire che i dati generati viene accuratamente controllato per il controllo qualità. I dati riportati in figura 8 dimostra che i test qPCR può essere molto robusti nella loro specificità, come il saggio # 13 (A13) o hanno un elevato livello di variabilità che possono portare a variazioni tecniche come test # 34 (A34).

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> 71378 </td><td></td></tr><tr><td> KCl </td><td> Sigma-Aldrich </td><td> 60128 </td><td></td></tr><tr><td> L&#39;acido piruvico </td><td> Sigma-Aldrich </td><td> P4562 </td><td></td></tr><tr><td> Hepes </td><td> Sigma-Aldrich </td><td> 54457 </td><td></td></tr><tr><td> MgCl 2 </td><td> Sigma-Aldrich </td><td> M1020 </td><td></td></tr><tr><td> NaH 2 PO 4 monobasico </td><td> Sigma-Aldrich </td><td> P9791 </td><td></td></tr><tr><td> Destrosio </td><td> Sigma-Aldrich </td><td> G6721 </td><td></td></tr><tr><td> NaOH </td><td> Sigma-Aldrich </td><td> 72068 </td><td></td></tr><tr><td> EGTA </td><td> Sigma-Aldrich </td><td> O3777 </td><td></td></tr><tr><td> CaCl 2 </td><td> Sigma-Aldrich </td><td> 21115 </td><td></td></tr><tr><td> Proteasi, Tipo XIV </td><td> Sigma-Aldrich </td><td> P5147 </td><td></td></tr><tr><td> Collagenase, Tipo I </td><td> Worthington </td><td> C9891 </td><td></td></tr><tr><td> 1x Sospensione tampone del DNA (10 mM Tris, pH 8.0, 0.1 mM EDTA) </td><td> TEKnova, </td><td> PN T0221 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> B8667 </td><td></td></tr><tr><td> Sodio pentobarbital </td><td> Henry Schein Vet. Alimentazione * </td><td> Contattare il fornitore per l&#39;ordinazione </td><td> * Deve essere acquistato attraverso un laboratorio veterinario o animale professionale </td></tr><tr><td> ExoSAP IT </td><td> Affymetrix / USB </td><td> 78201 </td><td></td></tr><tr><td> CellsDirect 2x Mix RXN </td><td> Invitrogen </td><td> 11737-030 </td><td></td></tr><tr><td> SuperScript III RT Platinum Taq Mix </td><td> Invitrogen </td><td> 10928-042 </td><td></td></tr><tr><td> RT-STA Primer Mix </td><td> IDT </td><td> Costume </td><td></td></tr><tr><td> Nucleasi acqua gratis </td><td> Sigma-Aldrich </td><td> W4502 </td><td></td></tr><tr><td> WTA2 </td><td> Sigma-Aldrich </td><td> WTA2-50rxn </td><td></td></tr><tr><td> QIAquick PCR kit di purificazione </td><td> Qiagen </td><td> 28104 </td><td></td></tr><tr><td> Qiacube </td><td> Qiagen </td><td> 9001292 </td><td></td></tr><tr><td> NanoDrop Spettrofotometro </td><td>NanoDrop </td><td> 2000c </td><td></td></tr><tr><td> Bioanalyzer DNA 7500 Kit </td><td> Agilent </td><td> 7500 kit </td><td></td></tr><tr><td> Un colore kit etichettatura </td><td> Roche-NimbleGen </td><td> 5223555001 </td><td></td></tr><tr><td> Mus musculus 12x135k serie </td><td> Roche-NimbleGen </td><td> 5543797001 </td><td></td></tr><tr><td> GenePix 4200A Scanner </td><td> Molecular Devices </td><td> 4200A </td><td></td></tr><tr><td> TransPlex Completa intero kit di amplificazione trascrittoma (WTA2 Kit) </td><td> Sigma-Aldrich </td><td> WTA2-50RXN </td><td></td></tr></tbody></table>

single cell transcriptional profiling of adult mouse cardiomyocytes

Mentre numerosi studi hanno esaminato i cambiamenti di espressione genica da omogenati di tessuto cardiaco, questo impedisce studiando la variazione inerente stocastica tra le cellule all&#39;interno di un tessuto. Isolamento delle popolazioni di cardiomiociti pura attraverso una perfusione collagenasi dei cuori del mouse facilita la generazione di microarray singola cella per tutta l&#39;espressione genica trascrittoma, o qPCR di obiettivi specifici utilizzando gli array nanofluidic. Descriviamo qui una procedura per esaminare le singole cellule profili di espressione genica dei cardiomiociti isolati dal cuore. Questo paradigma permette per la valutazione delle metriche di interesse che non dipendono dalla media (per esempio la varianza tra le cellule all&#39;interno di un tessuto) che non è possibile quando si utilizza tessuti tradizionali flussi di lavoro intero per la valutazione dell&#39;espressione genica (Figura 1). Abbiamo raggiunto l&#39;amplificazione robusta del trascrittoma microgrammi singola cellula cedimento di cDNA doppio filamento che facilita l&#39;uso di microarray su icellule ndividual. Nella procedura viene descritto l&#39;uso di array NimbleGen che sono stati selezionati per la loro facilità d&#39;uso e possibilità di personalizzare il loro design. In alternativa, un trascrittasi inversa - amplificazione del target specifico (RT-STA) di reazione, permette di qPCR di centinaia di obiettivi da parte nanofluidic PCR. Utilizzando uno di questi due approcci, è possibile esaminare la variabilità di espressione tra le cellule, così come l&#39;esame di profili di espressione di tipi di cellule rare all&#39;interno di un tessuto. Nel complesso, il singolo gene approccio cellula espressione permette la generazione di dati che sono potenzialmente in grado di identificare profili di espressione idiosincratica che sono tipicamente mediati in sede di esame l&#39;espressione di milioni di cellule da omogenati tipici generati dai tessuti interi.

Watch this Scientific Journal Video about Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes at JoVE.com

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Singoli profili di espressione delle cellule consente la dettagliata analisi di espressione genica di cellule singole. Noi descrivere i metodi per l&#39;isolamento dei cardiomiociti, e preparare il lisati risultante sia per microarray intero trascrittoma o qPCR di obiettivi specifici.

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

single-cell-transcriptional-profiling-of-adult-mouse-cardiomyocytes

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Biology

17.2K Views.  Buck Institute for Research on Aging. Singoli profili di espressione delle cellule consente la dettagliata analisi di espressione genica di cellule singole. Noi descrivere i metodi per l'isolamento dei cardiomiociti, e preparare il lisati risultante sia per microarray intero trascrittoma o qPCR di obiettivi specifici.

Video: Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolamento e Cultura della neonatali mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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Biologia

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolamento e fisiologica Analisi di mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolamento e Cultura del topo adulto Cardiomyocytes per Cell Signaling e<em&gt; In vitro</em&gt; Cardiaca Ipertrofia

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Laser-assistita Microdissection (LAM) come strumento per trascrizionale Profiling di tipi di cellule individuali

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Isolamento, cultura e trasduzione di topo adulto cardiomiociti

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

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 ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

Gocciolina basati su codici a barre singola cella trascrittomica dei tessuti dei mammiferi adulti

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Profiling dell'espressione genica specifica del tipo di cella nel fegato del mouse

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Isolamento, trasfezione e cultura a lungo termine di cardiomiociti per topi e ratti adulti

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Analisi della α-Actinin Network in Cardiomiociti derivati da iPSC umani utilizzando la microscopia di localizzazione a singola molecola

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...