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 improves with surgery. The oocyte is covered by a vitelline layer, a layer of follicle cells, and connective tissue containing the blood vessels21. The entire structure is termed the &#34;follicle.&#34;</li>
	<li>Place the lobes of the ovaries that are densely packed with the follicles containing the oocytes in sterile&#160;Barth&#39;s Saline7 (MBS) supplemented with penicillin and streptomycin (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES (pH&#160;7.5, NaOH), 100 &#181;g/mL penicillin, and 100 &#181;g/mL streptomycin).</li>
	<li>Single-out stage V-VI21 follicles. Hold the ovary lobe with forceps. Lower the platinum loop (Figure 1) over the follicles and gently withdraw the loop to disrupt the connective tissue containing the follicle from the ovary tissue. 
	NOTE: Stage V-VI21 follicles are characterized by their size (1.0 - 1.2 mm in diameter) and by the good contrast between the dark pigmented animal hemisphere and the yellowish vegetal hemisphere.</li>
	<li>Transfer only healthy-looking follicles (i.e.&#160;perfectly spherical and with intact pigmentation) to a 35 mm diameter Petri dish by means of a plastic Pasteur pipette (cut the back to an opening diameter of 1.5 mm).</li>
</ol>
2. Microinjection of mRNA into the Cytoplasm
Note: The microinjection system described here is derived from that reported by Kressmann and Birnstiel22.
<ol>
	<li>Prepare cRNAs from the respective cDNAs coding for the subunits of the &#945;4&#946;2&#948; GABAA receptor by in vitro transcription, the addition of a poly (A+) tail, and RNA quantification by gel electrophoresis23.</li>
	<li>Prepare microinjection pipettes from borosilicate glass capillaries (1.0 mm outer diameter (OD), 0.58 mm inner diameter (ID), 100 mm length) using a micropipette puller. Break off the tips of the glass capillaries under a microscope using a micromanipulator and microfilament to create a tip diameter of 12 - 15 &#181;m with a beveled tip.</li>
	<li>Backfill the microinjection pipettes with paraffin oil using a 10 mL syringe, and then mount the microinjection pipette onto a homebuilt microinjection apparatus (Figure 2). 
	NOTE: The homebuilt oil hydraulic injection apparatus consists of a grill motor controlled by a foot pedal switch. An additional switch changes the turning mode of the motor. This motor drives a micrometer screw (0.5 mm/turn) that advances or retracts the plunger of a 10 &#181;L glass syringe. The injection is at a speed of about 3 rpm and a 0.5 turn corresponds to a 50 nL injection into a follicle. The tip of the syringe is connected to thick-walled polytetrafluoroethylene tubing that is itself connected to the injection needle by a very short piece of Tygon tubing. The injection capillary is held by a drill chuck that is controlled by a micromanipulator. The entire system is filled with paraffin oil. Any air bubbles should be avoided, as they will impair the injection system. The setup is lit with a cold light source. A stereomicroscope is required for optical control. Follicles are visualized under cold light with a stereomicroscope (40X magnification). Performance of the injection setup is tested by the injection of radioactive tracer into Xenopus oocytes. A volume of 45 - 55 nL should be delivered per injection.</li>
	<li>Using a pipette with a sterile plastic tip, test the setup by placing a droplet of sterile water onto the clean inside of a moisture-resistant thermoplastic (2 x 2 cm). Immerse the tip of the injection pipette into this droplet, and then turn the motor to retract the plunger (negative pressure inside the injection pipette). 
	NOTE: Water should enter the pipette and form a visible interface with the oil.</li>
	<li>Retract the tip of the pipette from the droplet and apply positive pressure to the inside of the injection pipette. A water droplet should form at the tip of the injection pipette.</li>
	<li>Prepare for the injection of the follicles by lining them up with the vegetal poles pointing upwards in the interstices of a nylon mesh (G: 0.8 mm) glued to the bottom of a Petri dish (60 mm) covered with MBS.</li>
	<li>Dispense 2 &#181;L of mRNA using a pipette with a sterile plastic tip onto the clean inside of a thermoplastic. Take the mRNA up into the injection pipette by applying negative pressure to the inside of the injection pipette.</li>
	<li>Position the injection pipette over an individual follicle using the micromanipulator.</li>
	<li>Insert the injection needle into the center of the vegetal pole and inject 50 nL of mRNA at a flow rate of 0.6 &#181;L/min by applying positive pressure to the inside of the pipette. Wait 5 - 10 s before removing the injection pipette tip from the follicle to avoid mRNA escape. 
	NOTE: The mRNA should be injected into the vegetal (yellowish) pole to avoid injection into the nucleus, which is located in the animal hemisphere.</li>
	<li>Transfer the injected follicles to a new Petri dish (35 mm) filled with 2 mL of MBS. Place the dish into a wine cooler set at 18 &#176;C. Incubate the injected follicles for 1 - 7 d before recording, depending on the identity of the newly expressed protein.</li>
</ol>
3. Stripping of the Follicles (Figure 3)
NOTE: As mentioned above, the oocyte covered by a vitelline layer, follicle cells, and connective tissue, which contains the blood vessels24, is known as a &#34;follicle.&#34; All layers except for the vitelline layer, which provides mechanical stability without preventing the access of solutions to the cell surface, must be removed before electrophysiological experiments. The follicle without the surrounding cell layers has previously been termed a &#34;denuded&#34; oocyte25. This step is usually performed on the same day as the electrophysiological experiments.
<ol>
	<li>Transfer ten injected follicles to a borosilicate glass tube containing 0.5 mL of MBS, 1 mg/mL collagenase, and 0.1 mg/mL trypsin inhibitor. Immerse the tube in a water bath maintained at 36 &#176;C. Incubate the follicles for 20 min and shake the tubes occasionally.</li>
	<li>Rinse the follicles by transferring them to a second tube containing 1 mL of MBS at RT. Leave them in the tube for about 10 s.</li>
	<li>Transfer the follicles to a third tube containing 0.5 mL of doubly concentrated MBS containing 4 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA), and incubate them for 4 min at RT. Shake the tube occasionally. 
	NOTE: The hypertonic solution (doubly concentrated MBS) is used to induce shrinkage of the oocytes within follicle cell layers, thereby detaching follicular cell layers from the oocyte; EGTA is added to inhibit collagenase activity.</li>
	<li>Rinse the follicles by transferring them to a second tube containing 1 mL of MBS at RT. Leave them in the tube for about 10 s.</li>
	<li>Transfer the oocytes to a Petri dish (35 mm diameter) containing 2 mL of MBS. Under a stereomicroscope, separate the outer envelopes from the follicles by simply pushing the naked oocyte away using the platinum loop. 
	NOTE: The outer envelopes of the oocytes stick firmly to the culture dish and can be easily separated from the oocytes. Some oocytes will spontaneously lose their outer envelopes and will be recovered as denuded oocytes. This procedure results in a relatively stable preparation that keeps the spherical appearance of the oocytes.</li>
</ol>
4. Fast Solution Change around the Oocyte
<ol>
	<li>Perform a voltage clamp experiment, as described9,26.</li>
	<li>Change the solution of the gravity-fed perfusion system by turning the perfusion switches as required by the experiment.
	<ol>
		<li>Apply the perfusion solution at 6 mL/min through a glass capillary with an inner diameter of 1.35 mm, the mouth of which is placed about 0.4 mm from the surface of the oocyte, to allow fast changes in agonist concentration around the oocyte. 
		NOTE: The rate of change has been previously estimated as 70% in less than 0.5 s27.</li>
	</ol>
	</li>
</ol>

<ol>
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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" width="1467">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">NaCl</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">71380</td>
			<td style="width:428px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">P-9541</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">S6014</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4&#160;</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">M-1880</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:663px;">CaCl2&#160;</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">223560</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:663px;">Ca(NO3)2&#160;</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">C1396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">HEPES</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Penicilin/streptomycin</td>
			<td style="width:240px;">Gibco</td>
			<td style="width:136px;">15140-148</td>
			<td>100 &#956;g penicillin/ml and 100 &#956;g streptomycin/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Platinum wire loop</td>
			<td style="width:240px;">home-made</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller</td>
			<td style="width:240px;">Zeitz-Instruments GmBH</td>
			<td style="width:136px;">DMZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringe&#160;</td>
			<td style="width:240px;">Hamilton</td>
			<td style="width:136px;">80300</td>
			<td>10 &#956;l, Type 701N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thick walled polytetrafluoroethylene tubing</td>
			<td style="width:240px;">Labmarket GmBH</td>
			<td style="width:136px;"></td>
			<td>1.0 mm OD&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Paraffin oil</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">18512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Nylon net, gauge 0.8 mm&#160;</td>
			<td style="width:240px;">ZBF Z&#252;richer Beuteltuchfabrik AG</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Borosilicate glass tube&#160;</td>
			<td style="width:240px;">Corning</td>
			<td style="width:136px;">99445-12</td>
			<td>PYREX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Collagenase NB Standard Grade</td>
			<td style="width:240px;">SERVA</td>
			<td style="width:136px;">17454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor type I-S</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">T-9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">EGTA</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">E3389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Glass capillary</td>
			<td style="width:240px;">Jencons (Scientific ) LTD.</td>
			<td style="width:136px;">H15/10</td>
			<td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillary</td>
			<td style="width:240px;">Harvard Apparatus Limited</td>
			<td style="width:136px;">30-0019</td>
			<td>1.0 OD X&#160; 0.58 ID X 100 Length mm (for microinjection)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillary&#160;</td>
			<td style="width:240px;">Harvard Apparatus Limited</td>
			<td style="width:136px;">30-0044</td>
			<td>1.2 OD X 0.69 ID X 100 Length mm (for two-electrode voltage clamp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">&#947;-Aminobutyric acid (GABA)</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">A2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">3&#945;,21-Dihydroxy-5&#945;-pregnan-20-one (THDOC)</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">P2016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">grill motor</td>
			<td style="width:240px;">Faulhaber&#160;</td>
			<td style="width:136px;"></td>
			<td>DC micromotor Type 2230 with gear Type 22/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">micrometer screw</td>
			<td style="width:240px;">Kiener-Wittlin</td>
			<td style="width:136px;">10400</td>
			<td>TESA, AR 02.11201</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Sterile plastic transfer pipettes</td>
			<td style="width:240px;">Saint-Amand&#160; Mfg.</td>
			<td style="width:136px;">222-20S</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

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20.0K Views.  University of Bern. 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. 

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

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

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.

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

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

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.

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

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

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 by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

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

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

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