Name: xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments
Uploaded: 2016-12-31T15:00:00
Description: Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

Animal experiments have been approved by the local committee of the Canton Bern Kantonstierarzt, Kantonaler Veterin&#228;rdienst Bern (BE85/15).
1. Preparation of Xenopus Oocytes
<ol>
	<li>Maintain frogs (Xenopus laevis) on a 12 h/12 h light/dark cycle in water that is strictly kept at 20 &#176;C.</li>
	<li>Remove the lobes of the ovaries from female frogs9. 
	NOTE: The removal of the lobes is a stimulus for regeneration, and often the quality of the oocytes...

<ol>
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R., Marbaix, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+frog+eggs+and+oocytes+for+the+study+of+messenger+RNA+and+its+translation+in+living+cells.">Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells.</a> Nature. 233 (5316), 177-182 (1971).</li><li>Soreq, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biosynthesis+of+biologically+active+proteins+in+mRNA-microinjected+Xenopus+oocytes.">The biosynthesis of biologically active proteins in mRNA-microinjected Xenopus oocytes.</a> CRC Crit Rev Biochem. 18 (3), 199-238 (1985).</li><li>Sigel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Xenopus+oocytes+for+the+functional+expression+of+plasma+membrane+proteins.">Use of Xenopus oocytes for the functional expression of plasma membrane proteins.</a> J Membr Biol. 117 (3), 201-221 (1990).</li><li>Dascal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Xenopus+oocytes+for+the+study+of+ion+channels.">The use of Xenopus oocytes for the study of ion channels.</a> CRC Crit Rev Biochem. 22 (4), 317-387 (1987).</li><li>Sigel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+single+sodium+channels+translated+by+Xenopus+oocytes+after+injection+with+messenger+ribonucleic+acid.">Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid.</a> J Physiol. 386, 73-90 (1987).</li><li>Sigel, E., Minier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+oocyte:+system+for+the+study+of+functional+expression+and+modulation+of+proteins.">The Xenopus oocyte: system for the study of functional expression and modulation of proteins.</a> Mol Nutr Food Res. 49 (3), 228-234 (2005).</li><li>Colman, A., Hames, B. 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A., Baker, ., Wolfgang, W. a. l. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+oocyte+microinjection+and+ion-channel+expression.">Xenopus oocyte microinjection and ion-channel expression.</a> Patch-Clamp Applications and Protocols. , 259-305 (1995).</li><li>Maldifassi, M. C., Baur, R., Sigel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+sites+involved+in+modulation+of+the+GABA+receptor+channel+by+the+intravenous+anesthetics+propofol,+etomidate+and+pentobarbital.">Functional sites involved in modulation of the GABA receptor channel by the intravenous anesthetics propofol, etomidate and pentobarbital.</a> Neuropharmacology. 105, 207-214 (2016).</li><li>Maldifassi, M. C., Baur, R., Pierce, D., Nourmahnad, A., Forman, S. 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E., Gonzalez-Salgado, A., Butikofer, P., Maser, P., Sigel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heteromeric+potassium+channel+involved+in+the+modulation+of+the+plasma+membrane+potential+is+essential+for+the+survival+of+African+trypanosomes.">A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes.</a> FASEB J. 29 (8), 3228-3237 (2015).</li><li>Gonzalez-Salgado, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=myo-Inositol+uptake+is+essential+for+bulk+inositol+phospholipid+but+not+glycosylphosphatidylinositol+synthesis+in+Trypanosoma+brucei.">myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei.</a> J Biol Chem. 287 (16), 13313-13323 (2012).</li><li>Gonzalez-Salgado, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+brucei+Bloodstream+Forms+Depend+upon+Uptake+of+myo-Inositol+for+Golgi+Complex+Phosphatidylinositol+Synthesis+and+Normal+Cell+Growth.">Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth.</a> Eukaryot Cell. 14 (6), 616-624 (2015).</li><li>Wu, M., Gerhart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raising+Xenopus+in+the+laboratory.">Raising Xenopus in the laboratory.</a> Methods Cell Biol. 36, 3-18 (1991).</li><li>Gurdon, J. B., Wilt, F. H., Wessels, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+clawed+frogs.">American clawed frogs.</a> Methods in developmental biology. , 75-84 (1967).</li><li>Elsner, H. A., Honck, H. H., Willmann, F., Kreienkamp, H. J., Iglauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+quality+of+oocytes+from+Xenopus+laevis+used+in+laboratory+experiments:+prevention+by+use+of+antiseptic+surgical+technique+and+antibiotic+supplementation.">Poor quality of oocytes from Xenopus laevis used in laboratory experiments: prevention by use of antiseptic surgical technique and antibiotic supplementation.</a> Comp Med. 50 (2), 206-211 (2000).</li><li>Green, S. 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E., Grassmann, A., Loyter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birnstiel+M.L.+Surrogate+genetics.">Birnstiel M.L. Surrogate genetics.</a> Transfer of Cell Constituents into Eucaryotic Cells. , 388-407 (1980).</li><li>Middendorp, S. J., Maldifassi, M. C., Baur, R., Sigel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+modulation+of+synaptic+and+extrasynaptic+GABAA+receptors+by+an+antagonist+of+the+high+affinity+benzodiazepine+binding+site.">Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.</a> Neuropharmacology. 95, 459-467 (2015).</li><li>Dumont, J. N., Brummett, A. 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The methods described in this article deviate from those used traditionally7-9. It is standard to expose the lobes of the ovary to a 1 to 2 h collagenase treatment8; isolate undamaged, denuded oocytes; and inject them with mRNA using commercial injection devices. This classical procedure has the following drawbacks: 1) Oocytes are likely to be damaged by the long exposure to high concentrations of collagenase. 2) The unstable denuded oocytes must be stored until the experiment. 3) Denuded oocytes ar...

Xenopus oocytes are widely used as an expression system (References 2 - 3, and references therein). They are able to properly assemble and incorporate functionally active multisubunit proteins into their plasma membranes. Using this system, it is possible to functionally investigate membrane proteins alone or in combination with other proteins, in order to study the properties of mutated, chimeric, or concatenated proteins, and to screen potential drugs.
Advantages of using oocytes over other heterologous expression systems include the simple handling of the giant cells, the high proportion of cells expressing foreign genetic information, the simple control of the environment of the oocyte by means of bath perfusion, and the control of the membrane potential.
The drawback of this expression system is the seasonal variation observed in many laboratories17-20. The reason for this variation is far from clear. Additionally, the quality of oocytes is often observed to vary strongly. Traditional methods7-9 have included the isolation of ovary lobes, the exposure of ovary lobes to collagenase for some h, the selection of denuded oocytes, and the oocyte microinjection. Here, a number of alternative, fast procedures are reported that have allowed us to work with this expression system for more than 30 years with no seasonal variation and little variation in oocyte quality.
The modified and improved methods described here for the isolation of oocytes, microinjection with cRNA, and removal of follicular cell layers can be used for the expression of any protein of choice in the Xenopus oocyte. The very simple method for fast solution changes of the medium around the oocyte may be applied to the study of any ligand-gated ion channel and of carriers.

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xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus oocytes were mechanically singled out using a platinum loop (Figure 1). The oocytes were microinjected with mRNA coding for the GABAA receptor subunits &#945;4, &#946;2, &#948;, 0.5:0.5:2.5 fmol/oocyte (Figure 2). After 4 d, follicular cell layers were removed (Figure 3). Oocytes were voltage clamped at -80 mV and exposed to increasing concentrations of &#947;-aminobutyric acid (GABA) in t...

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

The overall goal of this procedure is to express new proteins, such as ion channels in Xenopus oocytes. This method can help answer key questions in any field that requires a functional expression system. The main advantages of the variations of this technique are the speed, reliability, and improved survival of the oocytes.This method can provide insight into functioning of ion channels. Additionally, it can be applied to the study of any other protein. We first had the idea for this method when we realized that classical methods resulted in a low rate of oocyte survival.After removing the lobes of the ovaries from female frogs according to the text protocol, place the lobes in sterile Modified Barth's Solution supplemented with penicillin and streptomycin. Cut open the lobes to allow access of the tissue to oxygen. To single out stage five to six follicles, use forceps to hold the ovary and then lower a platinum loop over the follicles and gently withdraw the loop to disrupt the connective tissue with the follicle from the ovary tissue.Sort out healthy-looking oocytes using the platinum loop. Use a plastic Pasteur pipette with the tip cut to a 1.5 millimeter diameter to transfer selected follicles to a 35 millimeter diameter Petri dish. After preparing cRNA's for the GABA A receptor and micro-injection pipettes according to the text protocol, use paraffin oil and a 10 milliliter syringe with a thin needle to backfill a micro-injection pipette.Then mount the pipette onto a home-built micro-injection apparatus. Using a pipette with a sterile plastic tip, place a droplet of mRNA solution onto the clean side of a piece of moisture-resistant thermoplastic. Then immerse the tip of the injection pipette into the droplet and turn on the motor to retract the plunger.mRNA solution should enter the pipette and form a visible interface with the oil. Next, retract the tip of the pipette from the droplet, and apply a positive pressure to the inside of the injection pipette. A droplet of mRNA solution should form at the tip of the injection pipette.Using a 60 millimeter Petri dish with nylon mesh glued to the bottom and covered with MBS for injection, line up the follicles with their vegetal poles pointing upwards. With the micromanipulator, position the injection pipette over an individual follicle. Insert the injection needle into the center of the vegetal pole and apply positive pressure to the inside of the pipette to inject 50 nanoliters of mRNA at a flow rate of 0.6 microliters per minute.Wait five to 10 seconds before removing the injection pipette tip from the follicle to avoid messenger RNA escape. Transfer injected follicles to a new Petri dish filled with two milliliters of MBS. Place the dish into a wine cooler set at 18 degrees Celsius.Incubate the injected follicles for one to seven days before recording, depending on the identity of the newly-expressed protein. To strip follicles, transfer ten injected follicles to a borosilicate glass tube containing 0.5 milliliters of MBS, one milligram per milliliter of collagenase, and 0.1 milligram per milliliter of trypsin inhibitor. Immerse the tube in a 36-degree Celsius water bath and incubate the follicles for 20 minutes.The temperature here is critical because one degree more may kill the oocytes. Rinse the follicles by transferring them to a second tube containing one milliliter of MBS at room temperature, and leave them in the tube for about ten seconds. Then transfer the follicles to a third tube containing 0.5 milliliters of doubly-concentrated MBS containing four millimolars EGTA, and incubate the samples at room temperature for four minutes, occasionally shaking the tubes.The incubation time of four minutes is critical to ensure follicular layers detach from the oocyte. Rinse the follicles by transferring them to another tube containing one milliliter of MBS at room temperature, and leave them in the tube for about 10 seconds. Transfer the oocytes to a 35 millimeter Petri dish containing two milliliters of MBS.Then, under a stereo microscope, use a platinum loop to separate the outer envelopes from the follicles by simply pushing the naked oocyte away. Store the denuded oocytes until use. In this experiment, mechanically singled-out Xenopus oocytes were micro-injected with mRNA coating for GABA A receptor sub-units.Then follicular cell layers were removed. The oocytes were voltage-clamped at negative-80 millivolts and exposed to increasing concentrations of GABA and the presence of THDOC, a potent positive allosteric modulator of the GABA A receptor. This panel shows elicited current amplitudes depending on the GABA concentrations, indicating the sensitivity of this sub-unit combination toward GABA.The equation used was I(c)Imax/n)where c is the concentration of GABA, the EC50 is the concentration of GABA in the presence of one micromolar THDOC, eliciting a half-maximal current amplitude. Imax is the maximal current amplitude. I is the current amplitude, and n is the Hill coefficient.The EC50 of the alpha four beta two delta GABA A receptor amounted to 0.41 0.12 micromolar and n was 0.76 0.04 Once mastered, folliculal cell layers can be removed in half an hour if performed properly. After it's development, this technique paved the way for researchers to improve protein expression procedure in Xenopus oocytes. After watching this video, you should have a good understanding of how to isolate Xenopus oocytes surrounded by follicular cell layers their micro-injection with mRNA and subsequent removal of follicular cell layers.

Research

JoVE Journal

Biology

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

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Microinjection of Xenopus Laevis Oocytes

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Visualizing RNA Localization in Xenopus Oocytes

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

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

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Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...