Unterstützt von Career Development Award von der Forschung zur Blindheit, NIH EY 019312, und nicht zweckgebundenen Zuschuss von Forschung Prevent Blindness und EY 02687 (Department of Ophthalmology &amp; Visual Sciences an der Washington University) zu verhindern.

Herstellung von Elektroden <ol><li> Machen Sie die Aufnahme Elektroden mit einer Mikropipette Abzieher und polieren Sie die Elektroden mit Heizdraht unter dem Mikroskop. Für Maus Kegel Single-Cell-Saug-Aufnahme wird der innere Durchmesser der Elektrodenspitze ca. 6-7 um. </li><li> Bereiten Referenzelektroden. Wiegen Sie 1% Agar in destilliertem Wasser. Schmelzen Sie die Agar-Lösung in heißen Wasserbad. Füllen Sie das Glas Pipetten (L = 100 mm, OD / ID = 1/0.75 mm) mit 1% Agar mit Spritze. Festigen der Agar bei Raumtemperatur für 10 Minuten. </li><li> Schneiden Sie die Pipetten in zwei Hälften mit einem Diamant-Messer. </li><li> Weichen Sie die Referenz-Elektroden in Bezug Lösung für mindestens 24 Stunden bei 4 ° C. </li></ol> Einrichten des Tests <ol start="5"><li> Dunkle anzupassen einer Maus in einem schwarzen Käfig über Nacht. </li><li> Bereiten Sie 100 mL Referenz-Lösung und 1 L Perfusionslösung (siehe unten) und filtrieren. </li><li> Man löst 0,1% BSA und 10 mM Glucose in 20 mL Referenzlösung. </li><li> Kalibrieren Sie die Lichtintensität der optischen Stimulator mit Photometer. </li><li> Montieren Sie die Aufnahme Kammer auf der Bühne des inversen Mikroskop. </li><li> Blase die Perfusionslösung mit 95% O 2 / 5% CO 2 inkubieren es in 40 ° C Wasserbad, um die Löslichkeit von CO 2 zu erhöhen. Die Lösung fließt aus der Flasche, um die Aufnahme Kammer durch einen keramischen Widerstand Erwärmen auf 36-38 ° C. Die Heizung sollte möglichst nahe an der Aufnahme Kammer wie möglich, idealerweise in der Phase des Mikroskops liegen. </li><li> Passen Sie den Durchflussregler für einen Volumenstrom von ca. 1-1,5 mL / min. </li><li> Füllen Sie die Aufnahme-Elektrode mit Referenz-Lösung. Legen Sie die Elektrode in einen Elektrodenhalter. Stellen Sie sicher, es gibt keine Luftblasen in der Elektrode. Montieren Sie die Halterung auf der headstage des Verstärkers. Schließen Sie die Halterung an eine Absaugvorrichtung, die von Verbindungsschlauch teilweise mit Mineralöl auf die Seite-Port des Elektrodenhalter gefüllt gebaut wird. Das andere Ende des Schlauches ist zu einem kleinen Reservoir von Mineralöl verbunden, an dem Druck durch den Mund eingesetzt werden. </li><li> Schließen Sie die Referenz-Elektrode mit dem anderen Pol der headstage. Binden Sie die Thermoelement (TC) auf die Referenz-Elektrode. Sicherstellen, dass die Elektrode und TC Tipps sind nahe beieinander, so dass die Temperaturanzeige so nah an der Zelle wie möglich getroffen werden. </li><li> Schalten Sie den Infrarot-Beleuchtung, um die Elektrode zu visualisieren. Die Elektrode wird durch Infrarot-Kamera an einen Monitor. Zentrum der Elektrode Bild auf das Gesichtsfeld und der Monitor mit einem Mikro-Manipulator. </li><li> Passen Sie die Position der Referenzelektrode Spitze nahe (2-4 mm), um die Aufnahme Elektrodenspitze. </li><li> Stellen Sie die DC-Spannung für die Heizung, so dass die Temperatur der Lösung beträgt etwa 36-38 ° C. </li><li> Run eine Dichtheitsprüfung, um den Widerstand der Aufnahme Elektrode überprüfen, in der Regel ca. 900 kOhm. </li></ol> Isolieren Sie die Maustaste Netzhaut <ol start="18"><li> Unter dim rotes Licht, euthanize Dunkeln angepasst Maus mit CO 2 und Genickbruch. Markieren Sie die Augäpfel mit der Spitze eines heißen Metalls Fackel durch Ausbrennen der dorsale Punkt der Sklera leicht. Entkernen die Augäpfel mit einer Schere. Alle nachfolgenden Verfahren sind unter Infrarot-Licht durchgeführt. </li><li> Hemisect die Augäpfel unter dem Mikroskop mit Infrarot-Beleuchtung und Bildwandler. </li><li> Entfernen Sie die Hornhaut und Linse. Entfernen Sie so viel von dem Glaskörper wie möglich. </li><li> Zur Aufnahme M-Kegel Antworten, entfernen Sie die ventralen Teil der Augenmuschel 4 mit einer Rasierklinge die Beurteilung aus dem Kauter zu markieren. </li><li> Peel die Netzhaut vom Pigmentepithel Schicht. </li><li> Flatten der Netzhaut und stelle sie auf den Boden der Petrischale mit 1-2 ml Referenzlösung gefüllt. </li><li> Scheiben der Netzhaut in kleine dünne Stücke mit einer Rasierklinge und inkubieren Sie die Zubereitung in einem lichtdichten Gehäuse mit reinem Sauerstoff gesättigt. </li></ol> Recordings <ol start="25"><li> Schalten Sie die Heizung vorübergehend. Schalten Sie die Perfusion Strömung zu einem Bypass-Schlauch, um die Kammer vor dem Austrocknen zu verhindern. </li><li> Lassen Sie die meisten der Lösung in die Kammer mit einem Stück Kimwipe Gewebe. Übertragen Sie die Netzhaut Federung, um die Aufnahme Kammer mit einer Glaspipette. </li><li> Warten Sie ca. 2 Minuten, bis die Netzhaut Scheiben nieder. Schalten Sie die Durchblutung wieder an. Schalten Sie die Heizung. </li><li> Schalten Sie Infrarot-Beleuchtung für die Vorbereitung zu visualisieren. Suche nach einem Stück der Netzhaut liegen auf der Kammer mit Photorezeptor äußeren Segmente herausragen (siehe Beispiel Abbildung). Draw inneren Segmente sanft in die Elektrode durch Absaugen. In dieser Konfiguration sollte mehrere Kerne und inneren Segmente in die Elektrode gezogen werden. Notieren Sie sich die Photoreaktion zu einem hellen Blitz zu testen, ob ein Kegel innere Segment in der Elektrode ist und ob die Zelle zu heilendein, die Beurteilung durch die Reaktion Kinetik und Amplitude. </li><li> Senken Sie den Mineral-Reservoir zu leichten Unterdruck an der Spitze der Elektrode zu helfen, halten Sie die innere Segment in der Elektrode gelten. </li><li> Rekord-Test-Flash-Reaktionen zwischen schwach und stark Intensität einmal eine gute Zelle gefunden wird. Testen Blitze aus einer kalibrierten optischen Stimulator 5 vorgesehen. Flash-Intensität und Wellenlänge sind durch eine Reihe von Neutralfilter und Schmalband-Interferenzfilter gesteuert. Testen Sie Blitzdauer (20 ms) wird vom Computer-gesteuerte Jalousien gesteuert. </li></ol> Lösungen <ol><li> Maus Perfusionslösung: 112,5 mM NaCl, 3,6 mM KCl, 2,4 mM MgCl 2, 1,2 mM CaCl 2, 10 mM HEPES (pH 7,4), 20 mM NaHCO 3, 3 mM Na-Succinat, 0,5 mM Na Glutamat, 0,02 mM EDTA, und 10 mM Glukose. </li><li> Maus Aufzeichnung Elektrode Lösung: 140 mM NaCl, 3,6 mM KCl, 2,4 mM MgCl 2, 1,2 mM CaCl 2, 3 mM HEPES (pH 7,4), 0,02 mM EDTA. </li></ol>

<ol>
	<li>Yau, K. W., Lamb, T. D., Baylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+fluctuations+in+membrane+current+of+single+toad+rod+outer+segments.">Light-induced fluctuations in membrane current of single toad rod outer segments.</a> Nature. 269, 78-80 (1977).</li><li>Nikonov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptors+of+Nrl+-/-+mice+coexpress+functional+S-+and+M-cone+opsins+having+distinct+inactivation+mechanisms.">Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.</a> J Gen Physiol. 125, 287-304 (2005).</li><li>Nikonov, S. S., Kholodenko, R., Lem, J., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+features+of+the+S-+and+M-cone+photoreceptors+of+wild-type+mice+from+single-cell+recordings.">Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.</a> J Gen Physiol. 127, 359-374 (2006).</li><li>Applebury, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+cone+photoreceptor:+a+single+cone+type+expresses+both+S+and+M+opsins+with+retinal+spatial+patterning.">The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.</a> Neuron. 27, 513-523 (2000).</li><li>Cornwall, M. C., Fein, A., MacNichol, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+that+underlie+bleaching+and+background+adaptation.">Cellular mechanisms that underlie bleaching and background adaptation.</a> J Gen Physiol. 96, 345-372 (1990).</li><li>Calvert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransduction+in+transgenic+mice+after+targeted+deletion+of+the+rod+transducin+alpha+-subunit.">Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.</a> Proc Natl Acad Sci U S A. 97, 13913-13918 (2000).</li></ol>

Einzel-Zell-Saug-Aufnahme von Sehzellen war 3 Jahrzehnten entwickelt. Es ermöglicht uns, trans-Membran Stromänderung durch Licht Stimulation ohne die Zellmembran überwinden induzierte aufzunehmen. Wegen der hohen Zell-Zell-Adhäsion, ist es schwierig, gesunde einzelnen Stäbchen und Zapfen von der Maus Netzhaut wie Amphibien Netzhaut zu isolieren und es ist schwer, einzelne Zapfen aufgrund der geringen Prozentsatz (3%) und kleine Größe zu finden. Die Innen-Segment-in (IS-in)-Aufzeichnungsverfahren überwindet diese...

single-cell suction recordings from mouse cone photoreceptors

Rod und Zapfen-Photorezeptoren in der Netzhaut sind verantwortlich für light detection. In der Dunkelheit sind zyklisch Nukleotid-gesteuerten (CNG)-Kanäle in das äußere Segment offen und ermöglichen Kationen stetig nach innen fließen durch die Membran, depolarisierende der Zelle. Belichtung löst die Schließung des CNG-Kanäle, die Blöcke der inneren Kationen Stromfluss und damit Ergebnisse in Zelle Hyperpolarisation. Basierend auf der Polarität von Photorezeptoren, wurde eine Saug-Aufnahme-Methode in den 1970er Jahren, dass, im Gegensatz zu den klassischen Patch-Clamp-Technik, nicht erforderlich ist Eindringen in die Plasmamembran entwickelt<sup&gt; 1</sup&gt;. Drawing das äußere Segment in ein eng anliegendes Glaspipette mit extrazellulären Lösung gefüllt ermöglicht die Aufzeichnung des aktuellen Veränderungen in einzelnen Zellen auf Test-Blitzbelichtung. Doch diese gut etablierte &quot;outer-Segment-in (OS-in)&quot; Saug-Aufnahme ist nicht geeignet für Maus Konus-Aufnahmen, da der geringe Anteil der Zapfen in der Netzhaut der Maus (3%) und die Schwierigkeiten bei der Identifizierung des Kegels äußeren Segmente. Kürzlich wurde eine innere-Segment-in (IS-in) Aufnahme-Konfiguration entwickelt, um das innere Segment / Kernregion des Photorezeptors in die Aufnahme Pipette ziehen<sup&gt; 2,3</sup&gt;. In diesem Video zeigen wir, wie man aus einzelnen Maus Kegel Photomorphosen mit Single-Cell-Saugelektrode aufzunehmen.

Watch this Scientific Journal Video about Single-cell Suction Recordings from Mouse Cone Photoreceptors at JoVE.com

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Wir werden zeigen, wie man Flash-Antworten von einfachen Mausklick Kegel mit einem Saug-Elektrode aufnehmen.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

single-cell-suction-recordings-from-mouse-cone-photoreceptors

Research

JoVE Journal

Biology

11.7K Aufrufe.  Washington University in St. Louis, School of Medicine. Wir werden zeigen, wie man Flash-Antworten von einfachen Mausklick Kegel mit einem Saug-Elektrode aufnehmen.

Serie: Single-cell Suction Recordings from Mouse Cone Photoreceptors

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Methoden für die Patch-Clamp Kapazität Recordings aus dem Kelch

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Forschung

Biologie

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

Whole Cell Recordings von Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Erstellung von Einzel-Zellsuspensionen aus Maus-Milz mit dem gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

Standardisierte Erstellung von Einzel-Zellsuspensionen aus Maus Lungengewebe mit dem gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Generierung von Einzel-Zellsuspensionen aus Maus Nervengewebe

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

Beziehen High Quality-RNA aus Einzel-Zellpopulationen in menschlichen Postmortem Gehirngewebe

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptional Profiling der erwachsenen Maus Kardiomyozyten

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

Generation der Mäuse aus induzierten pluripotenten Stammzellen

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

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

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

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

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

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

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Isolierung von Endothelzellen aus dem Lumen von Maus-Halsschlagadern für Einzelzell-Multi-Omics-Experimente

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...