Notre travail a été rendu possible par le soutien financier de McKnight Fonds de dotation pour les neurosciences (PAS), le National Institute on Drug Abuse (R01 DA019022; PAS) et un pré-doctorale NIH NRSA bourse pour PA (F31AA017042) de l&#39;Institut national sur les abus d&#39;alcool et l&#39;alcoolisme. Le mutant GIRK2-L257W a été fourni par le Dr Prafulla Aryal. Microscope à fluorescence et le parrainage aimablement fournies par les instruments Leica.

<ol>
	<li>Aryal, P., Dvir, H., Choe, S., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+alcohol+pocket+involved+in+GIRK+channel+activation.">A discrete alcohol pocket involved in GIRK channel activation.</a> Nature Neurosci. 12, 988-995 (2009).</li><li>Panchenko, V. A., Glasser, C. R., Mayer, 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=Structural+similarities+between+glutamate+receptor+channels+and+K++channels+examined+by+scanning+mutagenesis.">Structural similarities between glutamate receptor channels and K+ channels examined by scanning mutagenesis.</a> J Gen Phys. 117, 345-360 (2001).</li><li>Doyle, D. A., Cabral, M. o. r. a. i. s., Pfuetzner, J., Kuo, R. A., Gulbis, A., Cohen, J. M., Chait, S. L., &amp;amp, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+potassium+channel:+molecular+basis+of+K++conduction+and+selectivity.">The structure of the potassium channel: molecular basis of K+ conduction and selectivity.</a> Science. 280, 69-77 (1998).</li><li>Nishida, M., Cadene, M., Chait, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+Kir3.1-prokaryotic+Kir+channel+chimera.">Crystal structure of a Kir3.1-prokaryotic Kir channel chimera.</a> EMBO J. 26, 4005-4015 (2007).</li><li>Tao, X., Avalos, J. L., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+eukaryotic+strong+inward-rectifier+K++channel+Kir2.2+at+3.1+&amp;Aring;+resolution.">Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 &amp;Aring; resolution.</a> Science. 326, 1668-1674 (2009).</li></ol>

La mutagenèse dirigée est une approche classique pour étudier les relations structure-fonction des canaux ioniques. Il est basé sur la prémisse que, en changeant un résidu critique du canal ionique on devrait être capable d&#39;observer des changements prononcés dans la fonction de canal. En revanche, de nombreuses mutations non-critique de position peuvent être introduits sans perturbations majeures à la fonction de canal. Typiquement, le résidu en question est d&#39;abord muté en alanine, un petit non polaires des acides aminés, suivi par une mutation au tryptophane, la plus importante d&#39;acides aminés 2. Si le résidu est importante pour la fonction de canal, les deux mutations va changer l&#39;activité du canal. Si un résidu moins essentiel est muté, les effets de la mutation, en particulier à l&#39;alanine sont souvent anodins. Mutations tryptophane sont plus susceptibles de causer des changements à cause de problèmes stériques associés à la taille de tryptophane. Souvent, lorsque l&#39;information structurelle est manquant, la mutation systématique de l&#39;alanine ou de tryptophane dans un fragment de canal (appelé alanine ou tryptophane scan) peut être utilisé pour localiser les résidus critiques. Il est important de noter que les clones multiples avec différentes mutations ponctuelles peuvent être préparés en parallèle permettant de dépistage de résidus multiples dans des délais relativement courts. Récemment, plusieurs structures cristallines des canaux sont devenus disponibles 3,4,5. Ils peuvent être utilisés pour proposer potentiels résidus clés pour être testés en utilisant l&#39;approche décrite dans ce protocole. Les mesures sur le canal de type sauvage doit toujours accompagner la mesure d&#39;un canal de mutant pour servir de contrôle positif pour s&#39;assurer que les expériences de transfection et électrophysiologiques ont été effectués correctement. Des mutations ponctuelles peuvent conduire à un manque d&#39;un courant fonctionnelle, un projet similaire en cours à la régulation de type sauvage ou altéré de l&#39;activité du canal. Manque de courant mesurable peut être dû au trafic de pliage ou modifié incorrecte. Dans un tel cas il peut être utile pour savoir si le canal trafics correctement à la surface en utilisant un dosage biotinylation ou immunofluorescence contre le tag extracellulaire (sans perméabilisation cellulaire) faire la distinction entre non-fonctionnelles des canaux sur la membrane plasmique et des canaux de retenue à l&#39;intérieur de la cellule . Enfin, il est conseillé d&#39;étudier non seulement l&#39;activité basale du canal, mais aussi les conséquences de la mutation sur la régulation des canaux. Différents mécanismes de modulation de la fonction de canal pourrait être étudiée en fonction du canal ionique d&#39;intérêt, y compris la régulation par les protéines G, PIP2, des ions, la concentration en ATP, la phosphorylation (en utilisant la tyrosine, sérine thréonine ou mutation), l&#39;ubiquitination ou sumoylation (en utilisant la mutation lysine) et ouvreurs de canaux directs ou bloquants.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"> 
 
 <div>
 <table border="1">
 <thead>
 <tr>
 <td>Material Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comment</td>
 </tr>
 </thead>
 <tbody>
 
<tr>
<td>Chemicals</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Varies</td>
<td>&nbsp;</td>
<tr>
<td>Mini Prep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>27104</td>
<td>&nbsp;</td>
<tr>
<td>Radiacwash</td>
<td>&nbsp;</td>
<td>Biodex</td>
<td>005-100</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>CB40210</td>
<td>Poly-L-lysine can also be used</td>
</tr>
<tr>
<td>Maxi Prep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12263</td>
<td>&nbsp;</td>
<tr>
<td>QuickChange XL II Site Directed Mutagenesis Kit</td>
<td>&nbsp;</td>
<td>Stratagene</td>
<td>200512-5</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-027</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips &#x02C6;&#x2026; 12 mm</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>CS-12R</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Glass Tubing Borosilicate-standard wall</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>G150-3</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dmi3000-b/" target="_blank">Inverted fluorescence microscope</a></td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMI3000 B</td>
<td>&nbsp;</td>
<tr>
<td>Electrophysiology</td>
<td>&nbsp;</td>
<td>Nikon/ Leica/ Zeiss</td>
<td>Any inverted DIC (Hoffmann) microscope with fluorescence</td>
<td>&nbsp;</td>
<tr>
<td>Amplifier</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Axon Axopatch 200B amplifier</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion system</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>VC-6 Perfusion MM-6</td>
<td>&nbsp;</td>
<tr>
<td>Digitizer</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Digidata 1320 digitizer</td>
<td>&nbsp;</td>
<tr>
<td>Manipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Manipulator: MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Electrode Puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC10 vertical electrode puller</td>
<td>&nbsp;</td>
<tr>
<td>Isolation Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing Corporation</td>
<td>Air table</td>
<td>&nbsp;</td> </tbody>
 </table>
 </div>

mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells

Nous allons démontrer comment étudier les effets fonctionnels de l&#39;introduction d&#39;une mutation ponctuelle dans un canal ionique. Nous étudions la protéine G-dépendants de potassium intérieurement rectifier (dénommé GIRK) canaux, qui sont importants pour la régulation de l&#39;excitabilité des neurones. Il ya quatre sous-unités différentes de mammifères canal GIRK (GIRK1-GIRK4) - nous nous concentrons sur GIRK2 parce qu&#39;il forme un homotétramère. La stimulation de différents types de G récepteurs couplés aux protéines (RCPG), tels que le récepteur muscarinique (M2R), conduit à l&#39;activation des canaux GIRK. L&#39;alcool a également active directement les chaînes GIRK. Nous allons montrer comment muter un acide aminé par changer spécifiquement un ou plusieurs nucléotides dans l&#39;ADNc pour le canal GIRK. Cette séquence mutée ADNc sera amplifié dans les bactéries, purifié, et la présence de la mutation ponctuelle sera confirmée par séquençage de l&#39;ADN. L&#39;ADNc pour les chaînes de GIRK muté et de type sauvage sera transfecté dans des cellules embryonnaires humaines de rein HEK293T cultivées in vitro. Enfin, la cellule entière de patch-clamp électrophysiologie seront utilisés pour étudier les courants macroscopiques de potassium par les canaux exprimé ectopiquement GIRK de type sauvage ou muté. Dans cette expérience, nous allons examiner l&#39;effet d&#39;une mutation dans le L257W GIRK2 canaux sur l&#39;activation M2R-dépendants et alcoolo-dépendants.

Watch this Scientific Journal Video about Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells at JoVE.com

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

Nous allons démontrer comment étudier l&#39;effet d&#39;une mutation ponctuelle unique sur la fonction d&#39;un canal ionique.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

mutagenesis-functional-analysis-ion-channels-heterologously-expressed

Research

JoVE Journal

Biology

17.3K vues.  Salk Institute for Biological Studies. Nous allons démontrer comment étudier l'effet d'une mutation ponctuelle unique sur la fonction d'un canal ionique.

Vidéo: Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Enregistrement Patch Clamp des canaux ioniques exprimés dans les oocytes de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Recherche

Biologie

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulse-chase Analyse des chaînes de sucre N-glycoprotéines liées à des cellules de mammifères

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

L&#39;analyse de l&#39;ADN double-brin Break (ORD) de réparation dans les cellules de mammifères

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch Clamp et des techniques de perfusion pour étudier les canaux ioniques Exprimé en Xenopus Ovocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

Stratégie de transfection optimisés pour l&#39;expression et l&#39;enregistrement électrophysiologique des recombinants canaux ioniques voltage-dépendants présents au HEK-293T

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Analyse de la cinétique ARNm exportation nucléaire dans les cellules de mammifères par micro-injection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Protocoles de sélection mutagenèse et fonctionnelles pour l&#39;évolution dirigée de protéines dans les E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse de la situation du cycle cellulaire dans les cellules de mammifères

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolement et analyse fonctionnelle des mitochondries de cellules cultivées et de tissus de souris

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...