These observations suggest that the delayed cooperative activation of ms channels by voltage is not an intrinsic property of the channels but requires interactions between the patch of membrane and the patch pipette 7. This is intended as an introduction to patch clamp recording from xenopus laevis oocytes. Jan 10, 2011 to summarize, the mrna of bk channels is injected into xenopus laevis oocytes to express channel proteins on the oocyte membrane. The resulting ensemble currents are more consistent from well to well, and the success rate for each recording attempt is 95%. If youd like to speak to someone immediately, office contact information is. Aug 20, 2018 using the axoclamp 900a for twoelectrode voltage clamp of xenopus oocytes expressing ion channels t molecular devices. Patch clamp and perfusion techniques for studying ion. We report a new technique for improving the efficiency of singlechannel recording in the xenopus oocyte expression system. Using the axoclamp 900a for twoelectrode voltage clamp of xenopus oocytes expressing ion channels t molecular devices. Two techniques of oocyte preparation were compared. Moreover, multiple channels or conductance levels can be present at a time in the data that need to be quantified. Dec 07, 2015 see comment in pubmed commons below pflugers arch. Wholecell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. For experimentalists, the xenopus oocyte brings together in a single.
These patches may last for many minutes and are suitable for any electrophysiological study of ion channels expressed in xenopus oocytes, including singlechannel recordings. Since its development by sakmann and neher 1, 2, the patch clamp has become established as an. May 07, 2018 after a cdna clone for a particular channel has been isolated, it can be expressed in xenopus oocytes and studied using the patch clamp technique. Function of shaker potassium channels produced by cellfree.
Please complete and submit the form below and an adinstruments representative will aim to get in touch within one working day. Pdf patchclamp and perfusion techniques to study ion. Furthermore, xenopus oocytes are a leading system for studies of ion transport and channel physiology. This technique is used to measure the properties of expressed ion channels either in populations macropatch or individually singlechannel recording. Opusxpress 6000a is a semiautomated system for high throughput voltage clamp recording from xenopus oocytes.
Thank you for your interest in oocyte clamp amplifier. Patch clamp and perfusion techniques to study ion channels. A variety of techniques were used to mechanically stimulate oocytes during simultaneous voltage. Patch acquisition rates can be over 95% with healthy cells, clean pipettes, and an experienced researcher. In the population patch clamp ppc described herein, a single voltageclamp amplifier sums the wholecell currents from multiple cells at once, each sealed to a separate aperture in a planar substrate well. Explore the use of axoclamp 900a for twoelectrode voltageclamp of xenopus oocytes expressing ion channels. Electrical recordings of the mitochondrial calcium uniporter.
As a result, removal of the vitelline membrane for patchclamp. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter. Complete with hiclamp robot, usb video camera, desktop computer with installed hiclamp software, accessories, and. Patch clamp measurements on xenopus laevis oocytes. The european xenopus resource centre exrc is situated in portsmouth, united kingdom and provides tools and services to support researchers using xenopus models. Both species are fully aquatic, and are easy to maintain in captivity. Protease modulation of the activity of the epithelial.
Finally, some analysis techniques are unique to oo2 1 expression of ion channels in xenopus oocytes. Patchclamp and perfusion techniques to study ion channels. Imaging the activity and localization of single voltage. Automated voltageclamp screening system for xenopus laevis oocytes. 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.
One of the methods introduced by physiologists is the use of xenopus oocytes for expression of c. Oocytes injected with specific mrna, transcribed from. Improved preparation of xenopus oocytes for patchclamp. Xenopus laevis egg extracts the extract obtained from unfertilized xenopus eggs is a cellfree system which supplements and sometimes overcomes the use of other model systems. 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.
Two microelectrode voltage clamp tevc of xenopus oocytes. If youd like to speak to someone immediately, office contact information is available from the contact page. In the first technique i, standard methods were followed to prepare the oocytes for patch clamping 5, 7. These same features also make the oc725d ideal for clamping other large cells and cell structures such as squid axons. Manual defolliculation of xenopus oocytes uiowadblab. Xenopus is a genus of african frogs that are commonly known as the african clawed frogs. A simultaneous recording of video images of a membrane patch and currents through the same patch is shown in fig. To summarize, the mrna of bk channels is injected into xenopus laevis oocytes to express channel proteins on the oocyte membrane. Protease modulation of the activity of the epithelial sodium. There have been at least five major types of studies. Indeed, xenopus was the first vertebrate animal for which methods were developed to allow rapid analysis of gene function using misexpression by mrna injection. Xenopus oocytes, or when the patch pipettes are fabricated from or coated with soft glass 7.
Before patchclamp experiments, the oocytes were placed for 35 min at room temperature in a hypertonic medium 475 mosm with the following composition mm. Membranepipette interactions underlie delayed voltage. The female germ cells produce a primordial germ cell pgc, which then undergoes mitosis, forming oogonia. Induction of membrane blebs and vesicles from xenopus oocytes. Improved preparation of xenopus oocytes for patch clamp recording. Complete with hiclamp robot, usb video camera, desktop computer with installed hiclamp software, accessories, and consumables.
Ms channels could be activated by stretch in insideout, oncell, and outsideout patch. Patchclamp and perfusion techniques to study ion channels expressed in xenopus oocytes. The exrc depends on researchers to obtain and deposit xenopus transgenic and mutant lines, xenopus insitu hybridization clones, xenopus specific. Nih videocast molecular mechanisms of regulation of ion. To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive ms channel in xenopus oocytes, currents were recorded from oncell and excised patches of membrane with the patch clamp technique and from intact oocytes with the twoelectrode voltage clamp technique. Patch clamp electrophysiology, voltage clamp, action. Pdf the xenopus oocyte expression system is ideal for electrophysiological characterization of voltagedependent and liganddependent ion. Avoiding the formation of vesicles by patch excision from. Moreover, multiple channels or conductance levels can be present at a time in the data that need to be quantified to accurately. Patch clamp recordings from cellattached patches showed that serotonin, applied locally outside the patch, caused the opening of channels of. Mar 31, 2009 manual defolliculation of xenopus oocytes uiowadblab. 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. Here we present details of a system in which the twoelectrode voltage clamp technique is used to record electrical signals from xenopus oocytes under hydrostatic oil pressures of up to 60 mpa. 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.
Mechanically gated channel activity in cytoskeleton. 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. Cold spring harbor protocols, volume 2018, issue 4, april 2018, pages 258267 read more. Twoelectrode voltageclamp recordings in xenopus laevis oocytes. Experimental records of single molecules or ion channels from fluorescence microscopy and patchclamp electrophysiology often include highfrequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed. 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. Xenopus embryos for in vivo studies of human disease gene function. Patch clamp recording of ion channels expressed in xenopus. The oocyte plasma membrane is highly folded into macro and microvilli, thereby increasing the apparent surface area by a factor of 4. 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. The xenopus oocyte expression system is ideal for electrophysiological characterization of voltagedependent and. Introduction to xenopus xenbase a xenopus laevis and. For patch clamp experiments the vitelline layer has to be removed by placing the oocytes in hypertonic solution 20,21. We focus on techniques to maximize the quality of oocyte preparation and seal generation.
Electrical recordings of the mitochondrial calcium. May 39, 2020 at the national xenopus resource at the marine biological laboratory in woods hole, ma, usa. Ms channels could be activated by stretch in inside. Here, we describe the wellestablished technique of patch clamp recording from xenopus oocytes. Here, we present a protocol to study voltage and liganddependent activation of ion channels expressed in. Population patch clamp improves data consistency and.
Conventional methods of oocyte preparation adequate for twoelectrode voltageclamp often leave residual adhesions between the vitelline and plasma membranes. The actual surface area is reportedly between 18 mm2 22 and 20 mm2. Currents were recorded with the patch clamp technique from insideout patches obtained from xenopus oocytes expressing homotetrameric hcn2 channels. The singlechannel patch clamp method measures the kinetic behavior of single ion channel proteins as they undergo opening and closing conformational changes. Whole cell patch clamp recordings from morphologically digitimer ds2a. Frog food xenopus diets xenopus express has developed exclusive and complete xenopus diets through the years. Patchclamp recordings from cellattached patches showed that serotonin, applied locally outside the patch, caused the opening of.
Axoclamp 900a for twoelectrode voltageclamp of xenopus. Here we present details of a system in which the twoelectrode voltageclamp technique is used to record electrical signals from xenopus oocytes under hydrostatic oil pressures of up to 60 mpa. 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. In the first technique i, standard methods were followed to prepare the oocytes for patchclamping 5, 7.
An oocyte is a form of genetic material that can be collected for cryoconservation. Any of various aquatic, tongueless, clawed frogs of the genus xenopus of southern africa, certain species of which, especially the african clawed frog. Oocytes of the toad xenopus laevis are widely used as an expression system for ion channels, transporters, and receptors in drug development. The singlechannel patchclamp method measures the kinetic behavior of single ion channel proteins as they undergo opening and closing conformational changes. The oocyte is the cellular precursor for the development of specialized cells. Aug, 2004 the care of frogs, preparation of oocytes, solutions, chemicals voltage. Xenopus embryos are large and easily manipulated, and moreover, thousands of embryos can be obtained in a single day.
Xenopus oocytes are widely used as an in vivo system to study ion channels. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. For patchclamp experiments the vitelline layer has to be removed by placing the oocytes in hypertonic solution 20,21. Rating is available when the video has been rented. 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.
Using the axoclamp 900a for twoelectrode voltage clamp of. Your browser does not currently recognize any of the video formats. Thus, the ability to record wholecell currents from oocytes under high hydrostatic pressure would be very useful. Patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes. Currents were recorded with the patchclamp technique from insideout patches obtained from xenopus oocytes expressing homotetrameric hcn2 channels.
Twoelectrode voltage clamp of xenopus oocytes under high. Automated voltageclamp screening for xenopus oocytes. As a result, removal of the vitelline membrane for patch clamp recording produces microscopic damage to the. Two species of xenopus are regularly used by biologists, xenopus laevis and xenopus tropicalis. Nevertheless, the low throughput of manually performed.
Sequential twoelectrode voltage clamp tevc recording in oocytes, nondestructive usage of. Twoelectrode voltageclamp tevc recording in xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion. Voltage clamp recordings from xenopus oocytes request pdf. Formulated to our rigid specifications to provide laboratories and hobbyists with premium grade african clawed frog food. After a cdna clone for a particular channel has been isolated, it can be expressed in xenopus oocytes and studied using the patchclamp technique. Conventional methods of oocyte preparation adequate for twoelectrode voltage clamp often leave residual adhesions between the vitelline and plasma membranes. Xenopus definition of xenopus by the free dictionary. Voltageinduced membrane displacement in patch pipettes. 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. The care of frogs, preparation of oocytes, solutions, chemicals voltage. Your browser does not currently recognize any of the video formats available.
Roboocyte2 oocyte screening goes automatic youtube. The oc725d oocyte clamp is designed for twoelectrode, wholecell voltage clamping of xenopus oocytes. Apr 17, 2018 patch clamp and perfusion techniques to study ion channels expressed in xenopus oocytes. An oocyte is produced in the ovary during female gametogenesis.
Abstract we report a new technique for improving the efficiency of singlechannel recording in the xenopus oocyte expression system. Improved preparation of xenopus oocytes for patchclamp recording. Patch clamp and perfusion techniques for studying ion channels. Experimental records of single molecules or ion channels from fluorescence microscopy and patch clamp electrophysiology often include highfrequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed.
141 21 1341 693 183 879 740 1069 901 1 326 297 1527 968 1205 800 274 920 205 1337 377 350 47 1135 190 1089 47 190 88 70 1218 1440 619 1179 614