Supportato da Career Development Award da ricerca per prevenire la cecità, NIH concedere EY 019312, e sovvenzione illimitata di ricerca per prevenire la cecità e EY 02687 (Department of Ophthalmology &amp; Visual Sciences alla Washington University).

Fare elettrodi <ol><li> Rendere gli elettrodi di registrazione con un estrattore micropipetta e lucidare gli elettrodi con filamento di riscaldamento sotto il microscopio. Per la registrazione cono di aspirazione del mouse singola cellula, il diametro interno sulla punta dell&#39;elettrodo è di circa 6-7 micron. </li><li> Preparare elettrodi di riferimento. Pesare 1% agar-agar in acqua distillata. Sciogliete la soluzione agar in bagno d&#39;acqua calda. Riempire le pipette di vetro (L = 100 mm, OD / ID = 1/0.75 mm) con l&#39;1% agar con siringa. Solidificare l&#39;agar a temperatura ambiente per 10 minuti. </li><li> Tagliare le pipette a metà con un coltello di diamante. </li><li> Immergere gli elettrodi di riferimento nella soluzione di riferimento per almeno 24 ore a 4 ° C. </li></ol> Di iniziare l&#39;esperimento <ol start="5"><li> Scuro adattare un topo in una gabbia nera durante la notte. </li><li> La preparazione di 100 ml soluzione di riferimento e soluzione 1 perfusione L (vedi sotto) e filtrare la soluzione. </li><li> Sciogliere 0,1% BSA e 10 mM glucosio in 20 mL di soluzione di riferimento. </li><li> Calibrare l&#39;intensità della luce dello stimolatore ottico con fotometro. </li><li> Montare la camera di registrazione sul palco del microscopio invertito. </li><li> Bolla la soluzione di perfusione con il 95% O 2 / 5% di CO 2, incubare a 40 ° C acqua del bagno per aumentare la solubilità di CO 2. La soluzione fluisce dalla bottiglia alla camera di registrazione passa attraverso una resistenza ceramica riscaldamento a 36-38 ° C. Il riscaldatore deve essere posizionato più vicino alla camera di registrazione possibile, idealmente nella fase del microscopio. </li><li> Regolare il regolatore di flusso per una portata di circa 1-1,5 ml / min. </li><li> Riempire l&#39;elettrodo di registrazione con la soluzione di riferimento. Inserire l&#39;elettrodo in una porta elettrodo. Assicurarsi che non vi siano bolle in dell&#39;elettrodo. Montare il supporto sulla headstage dell&#39;amplificatore. Collegare il titolare di un dispositivo di aspirazione, che è costruito da collegamento di tubi parzialmente riempito con olio minerale al side-port del titolare elettrodo. L&#39;altra estremità del tubo è collegato ad un piccolo serbatoio di olio minerale, a cui la pressione può essere applicata per via orale. </li><li> Collegare l&#39;elettrodo di riferimento per l&#39;altro polo del headstage. Associare la coppia termica (TC) per l&#39;elettrodo di riferimento. Assicurarsi che le punte degli elettrodi e TC sono vicini tra loro in modo che la lettura della temperatura è presa il più vicino alla cella possibile. </li><li> Accendere l&#39;illuminazione a infrarossi per visualizzare l&#39;elettrodo. L&#39;elettrodo è vista da telecamera a infrarossi collegata a un monitor. Centrare l&#39;immagine elettrodo sul campo visivo e del monitor utilizzando un micro-manipolatore. </li><li> Regolare la posizione della punta stretta elettrodo di riferimento (2-4 mm) per l&#39;elettrodo di registrazione. </li><li> Regolare la tensione continua per il riscaldamento in modo che la temperatura della soluzione è di circa 36-38 ° C. </li><li> Eseguire un test di tenuta per verificare la resistenza di elettrodo di registrazione, di solito circa 900 kΩ. </li></ol> Isolare la retina del mouse <ol start="18"><li> Sotto fioca luce rossa, eutanasia il mouse adattato all&#39;oscurità con CO 2 e dislocazione cervicale. Segna i bulbi oculari usando la punta di una torcia metallo caldo da cauterizzare il punto più dorsale della sclera leggermente. Enucleare i bulbi oculari con le forbici. Tutte le procedure successive sono eseguite sotto la luce a infrarossi. </li><li> Hemisect i bulbi oculari sotto microscopio con illuminazione a infrarossi e convertitori di immagini. </li><li> Togliere la cornea e la lente. Rimuovere la maggior quantità di vitreo possibile. </li><li> Per registrare M-cono risposte, rimuovere la parte ventrale della conchiglia oculare 4 con una lametta a giudicare dal punto cauterizzazione. </li><li> Sbucciare la retina dallo strato dell&#39;epitelio pigmentato. </li><li> Appiattire la retina e posizionarlo sul fondo della scatola di Petri riempite con 1-2 mL di soluzione di riferimento. </li><li> Tagliate la retina in piccoli pezzi sottili con una lametta e incubare la preparazione in una scatola a tenuta di luce satura di ossigeno puro. </li></ol> Registrazioni <ol start="25"><li> Spegnere il riscaldatore temporaneamente. Accendere il flusso di perfusione ad una tubazione di bypass per evitare che la camera di essiccazione. </li><li> Scolate la maggior parte della soluzione nella camera con un pezzo di tessuto Kimwipe. Trasferire la sospensione retina alla camera di registrazione usando una pipetta di vetro. </li><li> Attendere circa 2 minuti fino a quando le fette retina stabilirsi. Accendere la perfusione indietro. Accendere il riscaldamento. </li><li> Accendere l&#39;illuminazione a infrarossi per visualizzare la preparazione. Ricerca di un pezzo di retina sdraiato sul da camera con segmenti esterni dei fotorecettori sporgenti (vedi figura esempio). Disegnare i segmenti interni dolcemente verso l&#39;elettrodo di aspirazione. In questa configurazione, i nuclei numerosi e segmenti interni deve essere aspirato nella dell&#39;elettrodo. Registrare il fotorisposta di un lampo luminoso per verificare se un segmento di cono interno è l&#39;elettrodo e se la cella è guariretua, a giudicare dalla risposta cinetica e ampiezza. </li><li> Abbassare il serbatoio minerale di applicare una leggera pressione negativa per la punta di un elettrodo per contribuire a tenere il segmento interno nella elettrodo. </li><li> Record di prova-flash risposte dim all&#39;intensità luminosa una volta una cella buona è stata trovata. Lampi di prova sono forniti da un calibrato stimolatore ottico 5. L&#39;intensità del flash e lunghezza d&#39;onda sono controllati da una serie di filtri a densità neutra e filtri di interferenza a banda stretta, rispettivamente. Test di durata del flash (20 ms) è controllato da computer-guidata persiane. </li></ol> Soluzioni <ol><li> Topo soluzione perfusione: 112,5 mM NaCl, 3.6 mM KCl, 2,4 mM MgCl 2, 1.2 mM CaCl 2, 10 HEPES mM (pH 7,4), 20 mM NaHCO 3, 3 mM Na succinato, 0,5 mM Na glutammato, 0.02 mM EDTA, e 10 mM glucosio. </li><li> Topo elettrodo soluzione di registrazione: 140 mM NaCl, 3.6 mM KCl, 2,4 mM MgCl 2, 1.2 mM CaCl 2, 3 HEPES mM (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>

Cella singola registrazione di aspirazione da fotorecettori è stato sviluppato 3 decenni fa. Essa ci permette di registrare trans-membrana variazione di corrente indotta dalla stimolazione luminosa senza penetrare la membrana cellulare. A causa della elevata adesione cellula-cellula, è difficile isolare sano singola asta e il cono di retina del mouse come retina anfibi ed è difficile trovare coni singoli a causa della bassa percentuale (3%) e piccole dimensioni. L&#39;interno-in-segmento (IS-in) metodo di registrazione supera questa difficoltà con la registrazione corrente dal segmento interno dei fotorecettori diversi, tra cui un cono potrebbero essere inclusi. Nel topo wild-type, la risposta dovrebbe essere la combinazione di coni e bastoncelli. Una luce di fondo costante può essere utilizzata per sopprimere le risposte asta. In questo video, abbiamo usato Tα-/ - retina, in cui aste non può rispondere alla stimolazione luminosa, in quanto mancano le α-subunità della proteina G trasducina 6. Pertanto, il fotorisposta è solo da coni.

single-cell suction recordings from mouse cone photoreceptors

Bastoncelli e coni della retina sono responsabili per la rilevazione della luce. Nel buio, nucleotidi ciclici-dipendenti (CNG) canali nel segmento esterno sono aperte e permettono il flusso di cationi costantemente verso l&#39;interno attraverso la membrana, la cellula depolarizzanti. Esposizione alla luce provoca la chiusura dei canali CNG, blocca il flusso di corrente verso l&#39;interno zione e, quindi, si traduce in iperpolarizzazione delle cellule. Sulla base della polarità di fotorecettori, un metodo di registrazione di aspirazione è stato sviluppato nel 1970 che, a differenza del classico patch-clamp, non necessita di penetrare la membrana plasmatica<sup&gt; 1</sup&gt;. Disegnare il segmento esterno in una pipetta di vetro stretto raccordo riempito con soluzione extracellulare permette la registrazione dei cambiamenti in atto nelle singole cellule su test-esposizione flash. Tuttavia, questa ben consolidata &quot;esterno-in-segmento (OS-in)&quot; registrazione di aspirazione non è adatto per le registrazioni del cono del mouse, a causa della bassa percentuale di coni nella retina del mouse (3%) e le difficoltà nell&#39;identificare il cono segmenti esterni. Recentemente, un interno-in-segmento (IS-in) la registrazione di configurazione è stato sviluppato per disegnare il segmento interno / regione nucleare del fotorecettore nella pipetta di registrazione<sup&gt; 2,3</sup&gt;. In questo video, ci mostrerà come registrare da fotorisposte singoli cono del mouse utilizzando una singola cella di elettrodi di aspirazione.

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

Mostreremo come registrare le risposte flash da coni del mouse utilizzando un elettrodo di aspirazione.

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

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

Nanyang Technological University

Research

JoVE Journal

Biology

11.7K Views.  Washington University in St. Louis, School of Medicine. Mostreremo come registrare le risposte flash da coni del mouse utilizzando un elettrodo di aspirazione.

Video: 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.

Metodi per la registrazioni Morsetto capacità patch dal Calyx

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

Ricerca

Biologia

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.

Registrazioni cella intero cervello di adulti 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.

Preparazione di Single-Cell Sospensioni da Spleen mouse con il dissociatore gentleMACS

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.

La preparazione standardizzata di Single-Cell Sospensioni da tessuto polmonare del mouse utilizzando il dissociatore gentleMACS

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.

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

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.

L&#39;ottenimento di RNA di alta qualità da popolazioni di cellule singole nei tessuti umani cervello postmortem

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.

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 ...

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.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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-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 ...

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.

Isolamento delle cellule endoteliali dal lume delle arterie carotidi di topo per esperimenti multi-omici a singola cellula

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