Name: Visualizing RNA Localization in Xenopus Oocytes (Video) | JoVE Journal
Uploaded: 2010-01-14T17:00:00.000+00:00
Duration: 7 min 6 s
Description: Learn RNA localization visualization in Xenopus oocytes using fluorescent labeling and confocal microscopy. This method enables real-time monitoring of RNA transport dynamics in living cells for developmental biology research.

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5 minutes

Our work on RNA localization is supported by a grant from the NIH (R01GM071049) to KLM.

Part 1: Transcription of Fluorescently Labeled mRNA.
<ol>
 <li>Linearize plasmid DNA containing the RNA localization element or other relevant sequence and resuspend at 1 μg/μl with DEPC-treated H2O. The DNA template must have upstream promoter sites for transcription by T7, SP6, or T3 RNA polymerase.</li>
 <li>Add the following reagents to a sterile 1.5mL tube:
 <table>
 <tbody>
 <tr><td>a. 10X Tx buffer (see M&amp;M)</td><td>2 μl</td></tr>
 <tr><td>b. 20X cap/NTP mix (see M&amp;M)</td><td>1 μl</td></tr>
 <tr><td>c. 1 mM Alexa Fluor 546-14-UTP (Invitrogen)</td><td>1 μl</td></tr>
 <tr><td>d. UTP, [α-32P](1 μCi/μl) (Perkin Elmer)</td><td>1 μl</td></tr>
 <tr><td>e. DEPC-H2O</td><td>11 μl</td></tr>
 <tr><td>f. 0.2 M DTT</td><td>1 μl</td></tr>
 <tr><td>g. RNasin (Promega)</td><td>1 μl</td></tr>
 <tr><td>h. linear template DNA</td><td>1 μl</td></tr>
 <tr><td>i. RNA Polymerase (Promega)</td><td>1 μl</td></tr>
 </tbody>
 </table>
 </li> 
 <li>Mix reagents gently and centrifuge briefly (10 sec. in a microcentrifuge).</li>
 <li>Incubate for 2-4 hours at 37° C, covering the tube with aluminum foil to prevent photobleaching of fluorophore.</li>
 <li>Add 1 μl 1 mg/ml RNase-free DNase (Promega) to degrade template DNA.</li>
 <li>Incubate 15 minutes at 37° C.</li>
 <li>Add 79 μl 20 mM EDTA (pH 8.0) to stop the reaction.</li>
 <li>Remove 1 μl to a separate tube, labeled as "input" and save.</li>
 <li>Spin reaction through a 1 ml G50 column (see Materials &amp; Methods), which will include unincorporated nucleotides while excluding full length mRNA.</li>
 <li>Concentrate the RNA by ethanol precipitation:
 <ol class="lalpha">
 <li>Add 250 μl 100% ethanol, 10 μl 7M CH3COONH4, 1 μl carrier RNA (yeast tRNA, 10 μg/μl) or 1 μl glycogen (20 mg/ml).</li>
 <li>Mix well and freeze until solid at -80° C for &gt;30 minutes or on dry ice for 15 minutes. </li>
 <li>Spin at maximum speed in microcentrifuge for 15 minutes, decant supernatant.</li>
 <li>Wash with 150 μl of 75% ethanol.</li>
 <li>Spin for 3 minutes at maximum speed in microcentrifuge, decant supernatant.</li>
 <li>Dry the pellet at 37° C for 3 minutes, ensuring that all ethanol has evaporated.</li>
 </ol>
 </li>
 <li>Resuspend in 11 μl DEPC-H2O and remove 1 μl to a separate tube, labeled as "incorporated".</li>
 <li>Determine the percent incorporation and dilute the RNA to 50 nM (see Materials &amp; Methods for calculation). </li>
 <li>The RNA should be frozen in 5 μl aliquots, for single use to avoid freeze / thaw cycles and can be stored for several weeks at -80° C.</li>
</ol>
Part 2: Preparing for Microinjection.
<ol>
 <li>RNA Preparation: Thaw an aliquot of RNA (50 nM), and denature the RNA at 70° C for 3-5 minutes. Spin for 10 minutes in room temperature microcentrifuge at maximum speed to remove any particulates, and keep on ice.</li>
 <li>Oocyte Preparation:
 <ol class="lalpha">
 <li>Surgically remove ovary from Xenopus laevis females (Nasco). </li>
 <li>Trim pieces of Xenopus laevis ovary into a 50 ml conical tube containing 25 ml of Collagenase solution (see Materials &amp; Methods). </li>
 <li>Shake gently at 18° C for 15 minutes, or until oocytes are visibly separated from ovary. Due to batch to batch variation, collagenase treatment time may vary and should be monitored carefully through visual inspection to ensure defoliculation. </li>
 <li>Allow oocytes to settle in tube, remove solution and wash the oocytes with MBSH buffer (see Materials &amp; Methods). </li>
 <li>Repeat wash twice more for a total of three washes. This oocyte isolation protocol is similar to that detailed in Cohen et al., 2009. </li>
 <li>Manually sort stage III/IV oocytes (Dumont, 1972) in MBSH buffer under a standard dissecting microscope. Stage III oocytes are completely opaque but white, while stage IV oocytes are slightly larger and speckled with pigment. Oocytes that are slightly transparent are too young and oocytes that fully pigmented or exhibit polarized pigment distribution are too old.</li>
 </ol>
 </li>
 <li>Needle Preparation: Pull and bevel needles to an outer diameter of ~ 0.05 mm. (We use Drummond Scientific 3.5 inch capillaries (order # 3-000-203-G/X), a Sutter Instrument Co. micropipette puller and a WPI Inc. beveller.)</li>
</ol>
Part 3: Microinjection
<ol>
 <li>Calibrate needle with DEPC-H2O to deliver 2 nl per injection. We front load our needle using a gas-driven microinjector (see Materials &amp; Methods), and calibrate drop size using a micrometer. </li>
 <li>Load RNA into needle.</li>
 <li>Place sorted oocytes into an injection dish containing MBSH buffer. We microinject on a dish with a layer of black foam rubber. The pale oocytes stand out well on a white background.</li>
 <li>Carefully inject each oocyte with 2 nl of RNA at 50 nM.</li>
 <li>Expel RNA, rinse needle with DEPC-H2O, and load next RNA for injection.</li>
</ol>
Part 4: Oocyte Culture
<ol>
 <li>Place oocytes in a well of a sterile 24 well plate (Sigma Aldrich). We culture up to a thousand oocytes per well. </li>
 <li>Remove buffer and replace with 400 μl Oocyte Culture Medium (OCM; see Materials &amp; Methods) per well. Place culture plate inside an air-tight plastic container with wet paper towel to maintain moist environment during culture.</li>
 <li>Incubate oocytes at 18° C for time points ranging between 8 and 48 hours.</li>
</ol>
Part 5: Fixation, Dehydration and Storage of Oocytes
<ol>
 <li>Sort out any dead oocytes and place surviving oocytes in glass vials. We routinely observe &gt;90% survival. </li>
 <li>Place oocytes in MEMFA fixative (see Materials &amp; Methods) and rock for 20 minutes. Protect the oocytes from light by covering with aluminum foil.</li>
 <li>Wash oocytes by removing fixative and replacing with equal volume of MBSH buffer. Repeat this wash once more for a total of two washes.</li>
 <li>Wash oocytes into anhydrous methanol:
 <ol class="lalpha">
 <li>Remove half of the volume, replace with methanol.</li>
 <li>Repeat step "a" twice.</li>
 <li>Remove all of the solution, replace with methanol.</li>
 <li>Repeat methanol wash.</li>
 </ol>
 </li>
 <li>Oocytes can be stored at -20° C until ready to image.</li>
</ol>
Part 6: Imaging RNA Localization by Confocal Microscopy
<ol>
 <li>Before imaging, add Murray s Clearing Medium (2:1 benzyl benzoate-benzyl alcohol) to a glass bottom FluoroDish (WPI Inc.) to cover the imaging surface. </li>
 <li>Carefully transfer the oocytes from methanol to FluoroDish, minimizing the volume of methanol transferred.</li>
 <li>Image on an inverted confocal microscope (We have successfully used a Zeiss LSM510 and a Leica TCS SP2).</li>
</ol>
<img src="/files/ftp_upload/1704/1704fig1.jpg" alt="Microscopy image of red blood cells illustrating membrane structure variations and fluorescence intensity." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1: Visualization of Subcellular RNA Localization by Microinjection of Fluorescently Labelled Transcripts.  A.  Following microinjection, RNA labeled with Alexa Fluor 546 is initially restricted to the nucleus. B. After eight hours of culture, a fluorescently labeled control RNA can be seen uniformly distributed in the cytoplasm of the oocyte. C. However, fluorescently labeled RNA containing sequences that recruit the transport machinery can be seen in the process of subcellular localization to the vegetal pole of the oocyte (towards bottom). Scale bar = 50 μm.

<ol><li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+Xenopus+laevis+oocytes%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li>
<li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oogenesis+in+Xenopus+laevis+%28Daudin%29.+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals%5BTitle%5D)+AND+%22J.+Morphol%22%5BJournal%5D)">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J. Morphol. 136, 153-179 (1972).</li>
<li>Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+transport+in+ovo%3A+Simultaneous+visualization+of+two+RNAs%5BTitle%5D)+AND+%22Mol+Reprod+Dev%22%5BJournal%5D)">RNA transport in ovo: Simultaneous visualization of two RNAs.</a> Mol Reprod Dev. 76, 1115-1115 (2009).</li>
<li>Gautreau, D., Cote, C. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.</a> Development. 124, 5013-5020 (1997).</li>
<li>Lewis, R. A., Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PTB%2FhnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.</a> Mol Cell Biol. 28, 678-6786 (2008).</li>
<li>Messitt, T. J., Gagnon, J. A., Kreiling, J. A., Pratt, C. A., Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.</a> Dev Cell. 15, 426-436 (2008).</li>
<li>Mowry, K. L., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.</a> Science. 255, 991-994 (1992).</li>
<li>Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li>
</ol>

Experiments using laboratory animals were performed in accordance with the guidelines and regulations set forth by Brown University.

Here we have presented a protocol for visualizing mRNA localization in Xenopus oocytes. This method, using fluorescently labeled RNA transcripts has higher signal to noise ratio than previously obtained with digoxigenin-labeled transcripts and is simpler and faster than in-situ based approaches (Mowry and Melton, 1992, Gautreau et al., 1997). Using this method, we can engineer RNA sequence mutations and rapidly test for in vivo function. Further, by using additional Alexa-UTP fluorophores, multiple RNA ...

A correction to the step 2 of Part 1 for article Visualizing RNA Localization in Xenopus Oocytes.

Erratum: Visualizing RNA Localization in Xenopus Oocytes

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;10X Tx buffer&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;60 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;400 mM Tris-HCl (pH 7.5)&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;20 mM spermidine-HCl&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;20x cap/NTP mix&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;10 mM CTP&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;10 mM ATP&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;9 mM UTP&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;2 mM GTP&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;20 mM G(ppp)G Cap Analog (New England Biolabs)&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;G-50 column&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;Hydrate 5 g Sephadex G-50 beads (Sigma Aldrich) in 100 ml deionized H&lt;sub&gt;2&lt;/sub&gt;O. DEPC-treat for 30 min. and autoclave. Store incomplete stock at room temperature. Before use, add the following RNase-free solutions:&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;0.5 ml 0.2 M EDTA&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;1 ml 1 M Tris pH 8.0&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;0.5 ml 20% SDS&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Store complete G-50 solution at 4 &amp;deg; C.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Remove and discard the plunger from a 3 ml syringe (BD Biosciences) and place the barrel of the syringe into a 15 ml conical tube (Corning). Plug the syringe with a small amount of glass wool (a plug about half the size of a penny).&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Swirl complete G-50 solution to resuspend beads.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Add 2 ml G-50 solution to the empty column.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Spin for 1 minute at 1,000 x &lt;em&gt;g&lt;/em&gt; in benchtop centrifuge.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Add 200 &amp;mu;l DEPC-treated deionized H&lt;sub&gt;2&lt;/sub&gt;O to each column. Spin.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Repeat wash twice more for a total of three washes.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Remove syringe barrel to a fresh 15 ml conical tube.&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Collagenase solution&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;75 mg collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt; (Sigma Aldrich)&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;25 ml 0.1 M KPO&lt;sub&gt;3&lt;/sub&gt;+ (pH 7.4)&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;MBSH buffer&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;88 mM NaCl&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;1 mM KCl&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;2.4 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;0.82 mM MgSO&lt;sub&gt;4&lt;/sub&gt; X 7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;0.33 mM Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt; X 4H&lt;sub&gt;2&lt;/sub&gt;O&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;0.41 mM CaCl&lt;sub&gt;2&lt;/sub&gt; X 6H&lt;sub&gt;2&lt;/sub&gt;O&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;10 mM HEPES (pH 7.6)&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Oocyte Culture Medium&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;50% L15 medium&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;15 mM HEPES (pH 7.6)&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;1 mg/ml insulin&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;100 mg/ml gentamicin&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;50 U/ml nystatin&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;50 U/ml penicillin&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;50 mg/ml streptomycin&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;MEMFA solution&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;0.1 M MOPS (pH 7.4)&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;2 mM EGTA&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;3.7% formaldehyde&lt;/li&gt; &lt;/ul&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Computing RNA yield&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ul&gt; &lt;li class=&quot;none&quot;&gt;Determine CPM in &quot;input&quot; and &quot;incorporated&quot; samples using a standard scintillation counter.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;incorporation = (&quot;incorporated&quot;) / (10 x &quot;input&quot;)&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Typical incorporation values range between ~0.03 and 0.10.&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Maximum theoretical yields for different polymerases: T7, T3, SP6 - 2.64 &amp;mu;g&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Reaction yield in &amp;mu;g = (maximum yield of polymerase used) X (incorporation)&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;Dilute RNA to 50 nM = (&amp;mu;g RNA) / 320 / (length of RNA in bases) / (5X10&lt;sup&gt;-8&lt;/sup&gt;)&lt;/li&gt; &lt;li class=&quot;none&quot;&gt;The reaction usually yields ~50-100 &amp;mu;l of RNA at 50 nM.&lt;/li&gt; &lt;/ul&gt;</td></tr></tbody></table>

visualizing rna localization in xenopus oocytes

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.

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 ...

In this procedure, fluorescently labeled RNA. Transcripts are injected into XUS Cytes and their localization is monitored. First, the RNA is labeled with fluorescent nucleotides.In an in vitro transcription reaction, the RNA is then loaded into a microinjection apparatus. The oocytes are placed on an injection dish and carefully micro injected. Finally, the oocytes are cultured to allow RNA localization to occur before imaging on the confocal microscope.Hi, I'm James Gagnan from the laboratory of Dr.Kimberly Mallory at the Department of Molecular Biology, cell biology and biochemistry at Brown University. Today we're going to show you a procedure for visualizing subcellular RNA localization in Xap oocytes. We use this procedure in our laboratory to study the RNA architecture and protein factors required for RNA localization during early vertebrate development.So let's get started. To begin this procedure, transcribe mRNA, which is both radioactively and fluorescently labeled as described in the accompanying written protocol. Add DS to degrade the template DNA, and then stop the reaction by adding EDTA.Remove unincorporated nucleotides and other small molecules by spinning through a G 50 column. The RNA is then concentrated by ethanol precipitation, washed and resuspended. After determining the concentration by scintillation counting, dilute the RNA to 50 nano molar and store in five microliter aliquots at minus 80 degrees Celsius.When ready to perform the microinjection thaw an Eloqua of the labeled RNA denature at 70 degrees Celsius for five minutes. Then spin for 10 minutes at maximum speed in a micro centrifuge at room temperature to remove particulates and keep on ice. Next, isolate the cytes trim pieces of xap post lavu ovary into a 50 milliliter conical two containing 25 milliliters of three milligram per milliliter.Collagenase in a 0.1 molar potassium phosphate buffer at pH 7.4. Shake gently at 18 degrees Celsius due to batch to batch variation. Collagenase treatment time may vary and should be monitored carefully through visual inspection.To ensure defoliation continue shaking for 15 minutes or until oocytes are visibly separated from ovary, allow the oocytes to settle to the bottom of the conical tube. Remove the solution and replace with MBSH buffer. Repeat this, wash twice more for a total of three washes.Next, manually sort stage three to four CYTES and MBSH buffer under a standard light microscope. Stage three cytes are completely opaque, but white. While stage four cytes are slightly larger and speckled with pigment.Cytes that are slightly transparent are too young and cytes that are fully pigmented or exhibit polarized pigment distribution are too old. Finally, prepare the needles for microinjection using three and a half inch capillaries pulling and beveling to make needles with an outer diameter of approximately 0.05 millimeters. Now that the mRNA oocytes and needles are ready, proceed to microinjection.Start by calibrating the needle with desy water to deliver two nanometers per injection injection.Front. Load the needle using a gas controlled micro injector and calibrate the drop size using a micrometer after calibration front load the RNA to be injected into the needle. Next place the sorted oocytes on an injection dish In MBSH buffer, the dish contains a layer of black foam rubber.The white cyte stand out well against a black background. Then carefully inject each oocyte with two liters of RNA at 50 nanomolar. Be sure to inject each oocyte only once and check the needle regularly for clogging when the injection is complete.Expel the remaining RNA, rinse the needle with desy water and load the next RNA for injection. After the injection transfer the injected cytes to a well of a sterile 24 well plate up to a thousand. CYTES can be cultured per well.Remove the buffer and replace it with 400 microliters of cyte culture medium per well. Then place the culture plate inside a plastic container with a wet paper towel to maintain a moist environment during culture, incubate oocytes at 18 degrees Celsius for time points ranging between eight and 48 hours after the desired incubation period. The oocytes can be prepared for microscopy after incubation of the injected oocytes.Sort out any dead oocytes and place surviving oocytes in glass vials. Survival of larger than 90%of the oocytes is routinely observed. Replace the buffer with M-E-M-F-A fixative and rock the cytes for 20 minutes.Protect the oocytes from sunlight during fixation after the fixation period is complete. Wash the oocytes by removing the fixative and replacing with an equal volume of MBSH buffer. Repeat this wash once more for a total of two washes.Next, wash the oocytes into anhydrous methanol. First, remove half of the volume and replace with methanol. Repeat this two more times.Following these washes, remove all of the solution and replace with methanol. Repeat this methanol wash one more time. The fixed and dehydrated oocytes can be stored at minus 20 degrees Celsius until ready to image.Before imaging, add Marie's clearing medium to a glass bottom WPI fluoro dish to cover the imaging surface. Carefully transfer the oocytes from methanol to the fluoro dish, minimizing the volume of methanol transferred image the oocytes on an inverted confocal microscope, both a Zeiss LSM five 10 and aica TCSs P give good results. Now we'll show some representative images of subcellular RNA localization immediately following microinjection fluorescent RNA can be seen in the nucleus after eight hours of culture of fluorescently labeled control.RNA can be seen uniformly distributed in the cytoplasm of the oocyte. However, a fluorescently labeled RNA containing sequences that recruit the transport machinery can be seen in the process of subcellular localization to the vegetable pole of the oocyte. We've just shown you how to micro inject fluorescently labeled RNAs into zap cytes and observe RNA localization by confocal microscopy.When doing this procedure, it's important to use deci treated solutions to avoid RNAs contamination to culture, your cytes in OCM containing antibiotics, and to protect your cytes from sunlight whenever possible to avoid photo bleaching. So that's it. Thanks for watching and good luck with your experiments.

visualizing-rna-localization-in-xenopus-oocytes

Research

JoVE Journal

Biology

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.<br...

Microinjection of Xenopus Laevis Oocytes

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity.  An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells.  This protocol will help address questions related to spatial control of signaling.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparing Individual Drosophila Egg Chambers for Live Imaging

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

We describe methods to visualize nascent transcription during zygotic genome activation in single cells during early embryogenesis.

Visualizing Zygotic Genome Activation In Single Cells of Early Embryos

Learn RNA localization visualization in Xenopus oocytes using fluorescent labeling and confocal microscopy. This method enables real-time monitoring of RNA transport dynamics in living cells for developmental biology research.

Visualizing RNA Localization in Xenopus Oocytes

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