Name: visualizing rna localization in xenopus oocytes
Uploaded: 2010-01-14T17:00:00
Description: Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

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 &mu;g/&mu;l 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 &mu;l</td></tr>
 <tr><td>b. 20X cap/NTP mix (see M&amp;M)</td><td>1 &mu;l<...

<ol>
	<li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+transport+in+ovo:+Simultaneous+visualization+of+two+RNAs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTB/hnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin.">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li></ol>

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

10X Tx buffer
<ul>
<li class="none">60 mM MgCl2</li>
<li class="none">400 mM Tris-HCl (pH 7.5)</li>
<li class="none">20 mM spermidine-HCl</li>
</ul>
20x cap/NTP mix
<ul>
<li class="none">10 mM CTP</li>
<li class="none">10 mM ATP</li>
<li class="none">9 mM UTP</li>
<li class="none">2 mM GTP</li>
<li class="none">20 mM G(ppp)G Cap Analog (New England Biolabs)</li>
</ul>
G-50 column
<ul>
<li class="none">Hydrate 5 g Sephadex G-50 beads (Sigma Aldrich) in 100 ml deionized H2O. DEPC-treat for 30 min. and autoclave. Store incomplete stock at room temperature. Before use, add the following RNase-free solutions:</li>
<li class="none">0.5 ml 0.2 M EDTA</li>
<li class="none">1 ml 1 M Tris pH 8.0</li>
<li class="none">0.5 ml 20% SDS</li>
<li class="none">Store complete G-50 solution at 4&deg; C.</li>
<li class="none">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).</li>
<li class="none">Swirl complete G-50 solution to resuspend beads.</li>
<li class="none">Add 2 ml G-50 solution to the empty column.</li>
<li class="none">Spin for 1 minute at 1,000 x g in benchtop centrifuge.</li>
<li class="none">Add 200 &mu;l DEPC-treated deionized H2O to each column. Spin.</li>
<li class="none">Repeat wash twice more for a total of three washes.</li>
<li class="none">Remove syringe barrel to a fresh 15 ml conical tube.</li>
</ul>
Collagenase solution
<ul>
<li class="none">75 mg collagenase from Clostridium histolyticum (Sigma Aldrich)</li>
<li class="none">25 ml 0.1 M KPO3+ (pH 7.4)</li>
</ul>
MBSH buffer
<ul>
<li class="none">88 mM NaCl</li>
<li class="none">1 mM KCl</li>
<li class="none">2.4 mM NaHCO3</li>
<li class="none">0.82 mM MgSO4 X 7H2O</li>
<li class="none">0.33 mM Ca(NO3)2 X 4H2O</li>
<li class="none">0.41 mM CaCl2 X 6H2O</li>
<li class="none">10 mM HEPES (pH 7.6)</li>
</ul>
Oocyte Culture Medium
<ul>
<li class="none">50% L15 medium</li>
<li class="none">15 mM HEPES (pH 7.6)</li>
<li class="none">1 mg/ml insulin</li>
<li class="none">100 mg/ml gentamicin</li>
<li class="none">50 U/ml nystatin</li>
<li class="none">50 U/ml penicillin</li>
<li class="none">50 mg/ml streptomycin</li>
</ul>
MEMFA solution
<ul>
<li class="none">0.1 M MOPS (pH 7.4)</li>
<li class="none">2 mM EGTA</li>
<li class="none">1 mM MgSO4</li>
<li class="none">3.7% formaldehyde</li>
</ul>
Computing RNA yield
<ul>
<li class="none">Determine CPM in "input" and "incorporated" samples using a standard scintillation counter.</li>
<li class="none">incorporation = ("incorporated") / (10 x "input")</li>
<li class="none">Typical incorporation values range between ~0.03 and 0.10.</li>
<li class="none">Maximum theoretical yields for different polymerases: 
T7, T3, SP6 - 2.64 &mu;g</li>
<li class="none">Reaction yield in &mu;g = (maximum yield of polymerase used) X (incorporation)</li>
<li class="none">Dilute RNA to 50 nM = (&mu;g RNA) / 320 / (length of RNA in bases) / (5X10-8)</li>
<li class="none">The reaction usually yields ~50-100 &mu;l of RNA at 50 nM.</li>
</ul>

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.

Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

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.

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 ...

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