In patch clamp experiments, suction is used to attach a micropipette filled with electrolyte solution to the cell membrane. Be the first to characterize a newly discovered ion channel in the cornea of the eye. Apr 17, 2018 here, we present a protocol to study voltage and liganddependent activation of ion channels expressed in xenopus oocytes using patch clamp techniques designed to control both the membrane voltage and the intracellular solution. As a result, removal of the vitelline membrane for patch clamp recording produces microscopic damage to the. If the pond dries up in the rainy season, xenopus laevis may migrate long distances to another pond, maintaining hydration by the rains. The exrc supplies oocytes to many uk laboratories regularly and also to some on mainland europe. Patchclamp applications and protocols edition 1 by alan a. The female germ cells produce a primordial germ cell pgc, which then undergoes mitosis, forming oogonia.
Two microelectrode voltage clamp tevc of xenopus oocytes introduction the voltage clamp technique is a method that allows ion flow across the cell membrane to be measured as an electric current as the transmembrane potential is held under constant experimental control with a feedback amplifier. Xenopus oocytes are widely used as an in vivo system to study ion channels upon injection with mrna 8 and more recently, as a biological system for incell nmr 9. Oocytes of the toad xenopus laevis are widely used as an expression system for ion channels, transporters and receptors in drug development until now, the low throughput of electrophysiology has prevented its use for secondary functional screening of drug targets. In the cell model and with r s 0, inaccuracy of c m estimates was 1% under widely varying conditions r. Use of the axoporator 800a for singlecell electroporation for transfection and. Induction of membrane blebs and vesicles from xenopus oocytes. Xenopus oocytes serve as a standard heterologous expression system for the study of cloned ion channels. This image shows the single channel events recorded from an excised segment of xenopus frog oocyte membrane using the patch clamp technique which will be used in this project. Patch clamp accessories including headstages, cables, and electrode holders. Nevertheless, the low throughput of manually performed. Improved preparation of xenopus oocytes for patchclamp recording article pdf available in pflugers archiv european journal of physiology 4335.
This chapter describes various methods for electrophysiological measurements for studying for ion channels and transport systems in xenopus oocytes. Godfrey, phd1, and george e sanders, dvm2,3 husbandry and health of the african clawed frog, xenopus laevis, greatly influences the quality of oocytes produced. Axon instruments and accessories, patchclamp accessories. Xenopus oocyte microinjection and ionchannel expression. Patch clamp amplifiers from single channels to large macroscopic recordings. Automated systems for expression in xenopus oocytes. Oocytes injected with specific mrna, transcribed from. Furthermore, xenopus oocytes are a leading system for studies of ion transport and channel physiology. The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level. Patch clamp measurements on xenopus laevis oocytes. Dynamic properties of calciumactivated chloride currents in xenopus laevis oocytes.
In 1976, they used firepolished micropipets with a tip diameter of 35 pm to record currents from a small patch of the membranbe of sk etal muscles, thereby. The large size of these cells allows for relatively easy expression and recording of activity of exogenous ion channels together with neurotransmitter receptors andor various regulatory proteins using the whole. Aug, 2004 in particular, in xenopus oocytes the high mechano. This contributes to reducing the number of female xenopus used for oocyte injection experiments.
North america, conference, boston, ma may 04 may 08, 2020 more than 2,800 worldrenowned experts, visionaries, and influencers from top pharma, biotech, academic, and government institutions convene in boston each spring for pegs. An oocyte is produced in the ovary during female gametogenesis. Patch clamp our sales team has years of experience in electrophysiology, too. These same features also make the oc725d ideal for clamping other large cells and cell structures such as squid axons. Effect of water hardness on oocyte quality and embryo development in the african clawed frog xenopus laevis earl w. Twoelectrode voltage clamp tev of xenopus laevis oocytes is easily applied for the rapid screening of ion channel function, in particular in pharmacological experiments. The oc725d oocyte clamp is designed for twoelectrode, wholecell voltage clamping of xenopus oocytes. The experiments described aid the researcher in pursuing new areas of electrophysiology and using the patch clamp. They are either bundled with an oocyte clamp amplifier or a more comprehensive two electrode voltage clamp workstation, which provide clamping and amplification of measured signals from glass microelectrodessharp electrodes. Currents were recorded with the patch clamp technique from insideout patches obtained from xenopus oocytes expressing homotetrameric hcn2 channels. Wholecell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle.
Oocytes of the toad xenopus laevis are widely used as an expression system for ion channels, transporters, and receptors in drug development. Electric circuit equivalent of the voltage clamp of the cell membrane with series resistance r s. Two microelectrode voltage clamp tevc of xenopus oocytes introduction the voltage clamp technique is a method that allows ion flow across the cell membrane to be measured as an electric current as the transmembrane potential is held under constant experimental control with a feedback. Effect of water hardness on oocyte quality and embryo. Jan 30, 2018 here, we present a protocol to study voltage and liganddependent activation of ion channels expressed in xenopus oocytes using patch clamp techniques designed to control both the membrane voltage and the intracellular solution. As a result, removal of the vitelline membrane for patch clamp recording produces microscopic. Measurements can then be recorded via a powerlab and labchart data acqusition system, which is purchased separately. A big part of this success has been the ability to provide a comprehensive range of highquality consumables and accessories to address a breadth of applications. Electrophysiology data from experiments performed in xenopus oocytes can be found. Sakman were the first to monitor the opening and closing of single ion channels and membranes by conductance measurements. Ionic current of bk channels is recorded using patch clamp techniques. Voltage clamp recordings from xenopus oocytes request pdf. During the preparation of oocytes for patch clamp, the 510 min treatment of stripping solution would detach the vitelline membrane from the plasma membrane, which makes possible stripping the vitelline membrane.
Regionalspecific differences in nicotinic acetylcholine receptors nachrs were examined using the wholecell patch clamp technique in rat medial habenula mhb slices. Patch clamp recording of ion channels expressed in xenopus. During oogenesis, the oogonia become primary oocytes. Mean agonist potency profiles in the middle and ventral thirds of the mhb were similar. 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. They can help you in choosing the right system for your specific application, give you advice on the general setup and useful accessories. For the past 30 years, oocytes from xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment.
Axon instruments consumables and accessories will satisfy all patch clamp needs. Here, we present a protocol to study voltage and liganddependent activation of ion channels expressed in xenopus oocytes using patch clamp. Patch clamp electrophysiology, voltage clamp, action. An amplifier designed for twoelectrode wholecell voltage clamping of large cells xenopus oocytes and cell structures squid axons. The african clawed frog, xenopus laevis, has a long history as an experimental animal, going back to endocrinology and embryology studies in the early 1950s. Patchclamp and perfusion techniques to study ion channels. Bk channels are expressed in xenopus oocytes by injecting messenger rna. N2 xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins.
This is intended as an introduction to patch clamp recording from xenopus laevis oocytes. Manual defolliculation of xenopus oocytes uiowadblab. The itev 90 amplifier is a complete twoelectrode voltage and current clamp amplifier providing the option to add a third electrode. We report a new technique for improving the efficiency of singlechannel recording in the xenopus oocyte expression system. Voltage clamp techniques hebrew university of jerusalem. Xenopus borealis as an alternative source of oocytes for. Improved preparation of xenopus oocytes for patch clamp recording article pdf available in pflugers archiv european journal of physiology 4335. We injected xenopus laevis oocytes with crnas complementary rnas encoding several kcnk channels and measured their response using the twoelectrode voltage clamp technique. These systems are designed for twoelectrode, wholecell voltage clamping of large cells such as xenopus oocytes and cell structures such as squid axons. 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. Patch clamp methods and protocols surveys the typical patch clamp applications and advises scientists on identifying problems and selecting the best technique in each instance. Methods patients one hundred consecutive chronic nonmalignant pain patients from the multidisciplinary pain centre, the national hospital,copenhagen,denmark were included in the crosssectional study. Highthroughput electrophysiology with xenopus oocytes. The ideal series resistance of the patch pipette is between 11.
The intracellular solution during patch clamp recordings is controlled by a perfusion system. The patch pipettes were pulled p2000, sutter instrument, novato, ca, usa from quartz tubing science products gmbh, hofheim, germany with an outer diameter of 1. Fast kinetic parameters that are derived from tev measurements are therefore seldom comparable to the same parameters measured with the patch clamp technique. Improved preparation of xenopus oocytes for patchclamp. Using the axoclamp 900a for twoelectrode voltage clamp of xenopus oocytes expression ion channels to characterize conus snail venom actions dr. Cftr, investigated with the twoelectrode voltageclamp. Researchers will find patch clamp methods and protocols to be an invaluable aid in the design and execution of a wide variety of patch clamp. Mechanically gated channel activity in cytoskeleton. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. The portfolio of amplifiers includes axopatch 200b for ultra lownoise singlechannel recordings, multiclamp 700b for wholecell voltage clamp and highspeed current clamp recordings. Two microelectrode voltage clamp tevc of xenopus oocytes introduction the voltage clamp technique is used to measure ionic currents in response to precisely controlled changes in the transmembrane potential of an isolated cell. The axon instruments series of amplifiers provide bestinclass solutions for the entire range of patch clamp experiments.
For experimentalists, the xenopus oocyte brings together in a single cell the capacity to. Patch clamp and perfusion techniques for studying ion. At times the ponds that xenopus laevis is found in dry up, compelling it, in the dry season, to burrow into the mud, leaving a tunnel for air. An oocyte is a form of genetic material that can be collected for cryoconservation. Measurements of membrane capacitance cm in xenopus laevis oocytes offer unique experimental possibilities but are difficult to perform with current methods. What is the best you fed with the wrong person moment youve seen. Axon instruments patchclamp amplifiers molecular devices.
Patch clamping cells in sliced tissue preparations, f. This twoelectrode voltage clamping tevc amplifier features. T1 hypotonicity activates a native chloride current in xenopus oocytes. Two microelectrode voltage clamp tevc of xenopus oocytes. Patch clamp electrophysiology is used to study the electrical properties of excitable cells and ion channels. Pdf improved preparation of xenopus oocytes for patchclamp. In other words, it is an immature ovum, or egg cell. It covers vitelline membrane removal, formation of a gigaohm seal gigaseal, and the optional conversion of the patch to the outsideout topology. Patch acquisition rates can be over 95% with healthy cells, clean pipettes, and an experienced researcher. These patches may last for many minutes and are suitable for any electrophysiological study of ion channels expressed in xenopus oocytes, including singlechannel recordings. Once the responses were isolated, xenopus oocytes were then used in a second type of study as an assay system to isolate cdna clones encoding the proteins in. Hypotonicity activates a native chloride current in. Researchers use this method most commonly to investigate membrane structures expressed in xenopus oocytes.
Conventional methods of oocyte preparation adequate for twoelectrode voltage clamp often leave residual adhesions between the vitelline and plasma membranes. The majority of cells in the ventral two thirds of the mhb responded robustly to local pressure application of nachr agonists. Another disadvantage of whole oocytes is that their cytoplasmic content can not be controlled. The twoelectrode voltage clamp tevc technique is used to study properties of membrane proteins, especially ion channels. The xenopus oocyte expression system is ideal for electrophysiological characterization of voltagedependent and liganddependent ion channels because of its relatively low background of endogenous channels and the large size of the cell. Hypotonicity activates a native chloride current in xenopus. Reducing the extracellular osmolarity by 50% elicited a calciumindependent chloride current having an anion conductivity sequence identical with swellinginduced chloride currents observed in. This may lead to a significant variability of measurements from different oocytes. Aug, 2004 the care of frogs, preparation of oocytes, solutions, chemicals voltage.
Avoiding the formation of vesicles by patch excision from. Xenopus express has developed exclusive and complete xenopus diets through the years. Automated voltageclamp screening for xenopus oocytes. We have found it best to send oocytes out as dissected ovary lobes on wet ice but can provide defolliculated oocytes if necessary, albeit at extra cost. Xenopus is also a unique system for analyses of genome evolution and whole genome duplication in vertebrates, as different xenopus species form a ploidy series formed by interspecific hybridization. Several studies on coexpressed cftr and enac in oocytes have been published since 1995, suggesting that the activation of cftr inhibits enac. To improve cm measurements in the twoelectrode voltage clamp tevc mode, we developed a pairedramp protocol and tested its performance in a model circuit with tunable cm, membrane resistance rm, and series resistance rs and in. The first part of patchclamp applications and protocols presents modern developments associated with the techn ogy of. The dedicated design of the oc725d includes such features as high compliance voltage 180 v and unique bath clamp circuitry. Leuven, campus gasthuisberg, b3000 leuven, belgium. The oocyte is the cellular precursor for the development of specialized cells. Formulated to our rigid specifications to provide laboratories and hobbyists with premium grade african clawed frog food.
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