Körlük ve EY 02.687 (Washington Üniversitesi Göz Hastalıkları ve Görsel Bilimleri Bölümü) önleyin Araştırma Körlük, NIH hibe EY 019.312 ve sınırsız ödenekle Önlemek için Kariyer Geliştirme Ödülü Research tarafından desteklenir.

Elektrotlar yapma <ol><li> Mikropipet çektirmenin kullanarak kayıt elektrotları olun ve mikroskop altında ısıtma filament elektrotlar lehçe. Fare koni tek hücreli emme kayıt için, elektrot ucu iç çapı yaklaşık 6-7 mm. </li><li> Referans elektrot hazırlayın. Distile su içinde% 1 agar tartılır. Agar çözüm sıcak su banyosunda eritin. Şırınga kullanarak% 1 agar ile cam pipetler (L = 100 mm, OD / ID = 1/0.75 mm) doldurun. 10 dakika boyunca oda sıcaklığında agar katılaşmaya. </li><li> Pipetler bir elmas bıçak kullanarak parçaya kesin. </li><li> 4 en az 24 saat için referans çözüm referans elektrotlar Soak ° C. </li></ol> Denemenin kurulumu <ol start="5"><li> Karanlık bir gecede, siyah bir kafes içinde bir fare adapte. </li><li> 100 ml referans çözümü ve 1 L perfüzyon çözeltisi (aşağıya bakın) hazırlayın ve çözüm filtre. </li><li> % 0,1 BSA ve 10 mM Glukoz, 20 ml referans çözelti içinde çözülür. </li><li> Işık şiddetinin fotometre kullanarak optik stimülatörü kalibre edin. </li><li> Inverted mikroskop aşamasında kayıt odasının monte edin. </li><li> % 95 O 2 /% 5 CO 2 ile Bubble perfüzyon çözüm, CO 2 çözünürlüğü artırmak için 40 ° C su banyosunda inkübe edin. Çözüm şişe, 36-38 ile ısınma seramik direnç geçerek kayıt odası ° C akar Isıtıcı, mikroskop aşamasında ideal kayıt odasına mümkün olduğunca yakın olarak yer almalıdır. </li><li> 1-1.5 ml / dak akış hızı akış regülatörü ayarlayın. </li><li> Kayıt elektrot referans solüsyonu ile doldurun. Bir elektrot tutucu elektrot yerleştirin. Elektrot hiçbir baloncuklar olmadığından emin olun. Amplifikatör headstage sahibi monte edin. Elektrot tutucu yan port kısmen mineral yağ ile dolu tüp bağlayarak yerleşik bir emme cihazı, tutucu bağlayın. Hortumun diğer ucunu ağız yoluyla hangi basınç uygulanabilir, mineral yağ küçük bir rezervuar bağlı. </li><li> Referans elektrot headstage diğer direğe bağlayın. Isıl çift (TC), referans elektrot bağlayın. Elektrot ve TC ipuçları birbirine yakın sıcaklık okuma, mümkün olduğu kadar hücre yakın olarak alınır, böylece emin olun. </li><li> Elektrot görselleştirmek için kızılötesi aydınlatma açın. Elektrot bir monitöre bağlı kızılötesi kamera tarafından görüntülendi. Merkezi görme alanı ve mikro-manipülatör kullanarak monitör elektrot görüntü. </li><li> Referans elektrot kayıt elektrot ucu ucuna yakın (2-4 mm) konumunu ayarlayın. </li><li> Çözeltinin sıcaklığı 36-38 hakkında ° C olacak şekilde ısıtıcı DC gerilim ayarlayın </li><li> Normalde 900 kÊ hakkında, kayıt elektrot direnci kontrol etmek için bir sızdırmazlık testi çalıştırın. </li></ol> Fare retina Yalıtımlı <ol start="18"><li> Loş kırmızı ışık altında, CO 2 ve servikal dislokasyon ile karanlık adapte fare euthanize. Biraz sklerasına en dorsal noktası dağlama, sıcak metal meşale ucu kullanarak gözbebekleri işaretleyin. Makas kullanarak gözbebekleri aydınlatmak. Sonraki tüm işlemler, kızılötesi ışık altında yapılmaktadır. </li><li> Kızılötesi aydınlatma ve görüntü dönüştürücüler ile mikroskop altında gözbebekleri Hemisect. </li><li> Kornea ve lens çıkarın. Vitreus kadar mümkün olduğu kadar çıkarın. </li><li> M-koni yanıtları kaydetmek için, koter işareti yargılamak bir jilet ile Göz yuvası 4 ventral kısmı çıkarın. </li><li> Retina pigment epitel tabakası soyulur. </li><li> Retina düzleştirin ve 1-2 ml referans solüsyonu ile dolu Petri kabı alt üzerine yerleştirin. </li><li> Retinanın bir jilet kullanarak küçük ince parçalar halinde dilimleyin ve saf oksijen ile doymuş bir ışık geçirmez kutu hazırlanmasında kuluçkaya yatmaktadır. </li></ol> Kayıtlar <ol start="25"><li> Isıtıcı geçici olarak kapatın. Odasının kurumasını önlemek için bir by-pass boru perfüzyon akım anahtarı. </li><li> En Kimwipe dokusunun bir parçası kullanarak odasında çözüm boşaltın. Cam bir pipet kullanarak kayıt odasına retina süspansiyon aktarın. </li><li> Retina dilim yerleşmek kadar yaklaşık 2 dakika bekleyin. Perfüzyon geçin. Isıtıcıyı açın. </li><li> Hazırlık görselleştirmek için kızılötesi aydınlatma açın. Retinanın fotoreseptör dış segmentleri (örneğin şekle bakınız) yapışmasını odasına yatarken bir parça için ara. Emme ile elektrot içine yavaşça iç segmentleri çizin. Bu yapılandırmada, birkaç çekirdek ve iç bölümleri elektrot çizilmelidir. Bir koni iç segment hücre iyileşmesi olup olmadığını elektrot olup olmadığını test etmek için parlak bir flaş photoresponse KayıtSenin, cevap kinetiği ve genlik yargılamak. </li><li> Mineral deposu, yardımcı elektrot iç segment tutun için elektrot ucu hafif negatif basınç uygulamak için indirin. </li><li> Kayıt test flaş yanıtları için loş aydınlık şiddeti iyi bir hücre bir kez. Test yanıp söner kalibre edilmiş bir optik Stimülatörü 5 sağlanmaktadır. Flash yoğunluğu ve dalga boyu sırasıyla, bir dizi nötral yoğunluk filtreleri ve dar bant girişim filtreleri tarafından kontrol edilir. Test flaş süresi (20 ms), bilgisayar kontrollü kepenkler tarafından kontrol edilir. </li></ol> Çözümler <ol><li> Fare perfüzyon çözüm: 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 süksinat, Na, 0.5 mM Na glutamat, 0.02 mM EDTA ve 10 mM glukoz. </li><li> Fare kayıt elektrot çözüm: 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>

Fotoreseptör hücrelerinin Tek hücreli emme kayıt 3 yıl önce geliştirilmiştir. Bu hücre zarının delinmesi ışık stimülasyonu ile indüklenen trans-membran geçerli değişikliği kaydetmek için bize sağlar. Yüksek hücre-hücre adezyon Çünkü, amfibi retina gibi fare retinanın sağlıklı tek bir çubuk ve koni izole etmek zor ve düşük oranda (% 3) ve küçük boyutu nedeniyle bireysel konileri bulmak zordur. Iç kademeli (IS-in) kayıt yöntemi bir koni dahil olabilir, bunların arasında birkaç ...

single-cell suction recordings from mouse cone photoreceptors

Çubuk ve koni retinadaki fotoreseptör ışık algılama sorumludur. Karanlıkta, dış segmente siklik nükleotid kapılı (CNG) kanalları açıktır ve katyonların hücre depolarize zarından içeri doğru istikrarlı bir şekilde akmasına izin verin. Işığa maruz kalma CNG kanalları, bloklar içe katyon akım, ve böylece hücre hiperpolarizasyon sonuçları kapatılması tetikler. Fotoreseptörlerin polarite dayanarak, bir emme kayıt yöntemi klasik yama klemp tekniği aksine, plazma zarı delinmesi gerektirmez, 1970&#39;li yıllarda geliştirilen<sup&gt; 1</sup&gt;. Ekstrasellüler çözümü ile dolu sıkı oturan bir cam pipet içine dış segmente Çizim test flaş pozlama üzerine tek tek hücrelerin içinde güncel değişiklikler kayıt sağlar. Ancak, bu köklü &quot;dış segment (OS)&quot; emme kayıt fare retinanın koni düşük oranda (% 3) ve koni belirlenmesinde zorluklar nedeniyle, fare koni kayıtları için uygun değildir dış segmentleri. Son zamanlarda, bir iç-segment (IS) kayıt yapılandırma kayıt pipet içine iç segment / fotoreseptör nükleer bölge çizmek için geliştirilmiştir<sup&gt; 2,3</sup&gt;. Bu video, tek hücreli emme elektrot kullanarak bireysel fare koni photoresponses nasıl kayıt gösterecektir.

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

Biz bir emme elektrot kullanarak tek bir fare kozalakları flaş cevaplarını kaydetmek için nasıl gösterecektir.

Tek hücreli fare Koni fotoreseptörlerin Emiş Kayıtlar

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 Görüntüleme.  Washington University in St. Louis, School of Medicine. Biz bir emme elektrot kullanarak tek bir fare kozalakları flaş cevaplarını kaydetmek için nasıl gösterecektir.

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.

Kaliks Patch Kelepçe Kapasitans Kayıtlar Yöntemleri

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

Biyoloji

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.

Yetişkin Drosophila Beyin Tüm Hücre Kayıtlar

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.

Fare Dalak gentleMACS Dissociator Tek Hücreli Cezalar hazırlanması

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.

Standardize Hazırlık gentleMACS Dissociator kullanarak Fare Akciğer Doku Tek Hücre Cezalar

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.

Fare Sinir Doku Hücre Tek Cezalar Üretimi

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.

İnsan Postmortem Beyin Dokusu Tek Hücre Popülasyonlar Yüksek Kalite RNA Alınması

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.

Yetişkin Fare kardiyomiyositlerde Tek Hücre transkripsiyonel Profilleme

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.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

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.

Tek Hücreli Multi-Omik Deneyler için Fare Karotis Arterlerinin Lümeninden Endotel Hücrelerinin İzolasyonu

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