Name: production of xenopus tropicalis egg extracts to identify microtubule-associated rnas
Uploaded: 2013-06-27T13:00:00
Duration: 10 min 1 s
Description: Watch this Scientific Journal Video about Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs at JoVE.com

1. Generation of X. tropicalis Eggs
All Xenopus tropicalis frogs are ordered from NASCO. Our frogs are housed in an Aquatic Habitats recirculating water system kept at 27 &deg;C. There are many options for water systems for care of X. tropicalis. Some good general information on this frog species can be found on the web sites of the Harland and Grainger labs (<a href="http://tropicalis.berkeley.edu/home/" target="_blank">http://tropicalis.berkeley.edu/home/</a>, <a href="http://www.faculty.virginia.edu/xtropicalis/" target="_blank">http://www.faculty.virginia.edu/xtropicalis/</a>). Our frogs are maintained in tankwater consisting of (0.4 g Ciclid Lake Salts, 0.6 g marine salt, 0.625 g NaHCO3 per liter of water, pH 7.0)20. This recipe results in a conductivity of ~1800 &mu;S, which is a high salinity for X. tropicalis. However, we have found that our frogs thrive in this environment and oocyte quality is improved. Alternative tankwater recipes can be found above at the resources listed for general X. tropicalis care.
<ol>
 <li>Frogs are injected with human Chorionic Gonadotropin (hCG) on three successive days to stimulate egg laying: First, prepare two concentrations of hCG solution. Resuspend 10,000 U of lyophilized hCG powder in 10 ml sterile, deionized H2O for a final concentration of 1,000 U/ml. Then, dilute 1 ml of 1,000 U/ml hCG solution in 9 ml H2O for a final concentration of 100 U/ml. Store both solutions at 4 &deg;C.</li>
 <li>On day 1, prepare 4-6 frogs for egg laying by injecting with hCG between 2:00-3:00 PM. Inject each frog in the dorsal lymph sac near the cloaca with 0.2 ml 100 U/ml hCG solution. Having the frogs fast during the subsequent two injections will minimize the amount of frog waste present during egg laying, but is optional.</li>
 <li>On day 2, inject the same frogs with 0.2 ml 100 U/ml hCG solution between 2:00-3:00 PM.</li>
 <li>On day 3, inject the same frogs with 0.2 ml 1,000 U/ml hCG solution, between 7:00-10:00 AM. Set up frogs to lay eggs: fill a 6-quart plastic bucket with fresh tankwater, add frogs and place in the dark at 25 &deg;C. After this injection, egg laying will begin after 4 hr and will be complete by 7 hr. Frogs should lay eggs in an environment that is maintained at a minimum of 25 &deg;C.</li>
 <li>Make extract solutions and have equipment ready immediately before collecting eggs.</li>
</ol>
20X MMR: 100 mM HEPES, pH 7.8; 2 mM EDTA pH 7.8; 2 M NaCl; 40 mM KCl; 20 mM MgCl2; 40 mM CaCl2. Autoclave and store at room temperature. Prepare 1 L of 1X MMR just prior to extract preparation.
10X XB: 100 mM HEPES, pH7.7; 10 mM MgCl2; 1 mM CaCl2; 1 M KCl; 500 mM sucrose. Autoclave and store at 4 &deg;C. Prepare 1 L of 1X XB just prior to extract preparation. Dejelly solution: Prepare 250 ml 3% cysteine solution in deionized H2O and pH to 7.8-8.0 with 10 N NaOH. Prepare just prior to extract preparation.
CSF-XB: take 200 ml of 1X XB and add 2 ml 0.5 M EGTA pH 7.7 and 200 &mu;l 1 M MgCl2. Prepare just prior to extract preparation.
CSF-XB+: take 50 ml of CSF-XB and add 50 &mu;l of LPC (10 mg/ml each stock of Leupeptin, Pepstatin, and Chymostatin in DMSO). Add 50 &mu;l Cytochalasin D (10 mg/ml in DMSO). Prepare just prior to extract preparation.
Prepare a 0.2% gelatin solution in deionized H2O, microwave to dissolve and filter sterilize. Store at room temperature.
Reserve 2 Beckman 2 x &frac12; inch Ultracentrifuge tubes.
Prepare two 15 ml glass round-bottomed centrifuge tubes with 0.5 ml of H2O in each to cushion the ultracentrifuge tube.
Make fire-polished glass Pasteur pipettes. Snap the end off of 5 &frac34; inch glass pipettes to expose a broad opening, and expose to flame to smooth the new exposed pipette tip.
<ol start="6">
 <li>Prepare a 500 ml glass beaker for storing eggs by swirling a 0.2% gelatin solution around to coat the walls of the beaker. Discard gelatin solution from beaker after use.</li>
 <li>Collect eggs from the plastic bucket used for laying 6-7 hr after the third injection on day 3. If desired, gently squeeze each frog once to get any remaining eggs. Wash eggs once with fresh tankwater and transfer to the 500 ml glass beaker coated with 0.2% gelatin solution.</li>
</ol>
2. Preparation of Extract from X. tropicalis Eggs
All steps of extract preparation can be performed at room temperature, approximately 25 &deg;C. Throughout the washes, it is important to keep the eggs submerged under liquid so that they remain wet. Exposure to air can cause the eggs to escape cell cycle arrest or lyse.
<ol>
 <li>Decant as much tankwater as possible while reserving enough liquid to keep the eggs wet. Tilt the beaker containing eggs to the side and add ~300 ml 1X MMR slowly to the wall of the beaker, so that physical agitation of the eggs is minimized. Let eggs settle, then decant off supernatant containing debris. X. tropicalis eggs are stringy at this step, so removal of activated eggs is done after dejellying. Repeat for a total of three 1X MMR washes.</li>
 <li>Dejelly the eggs. Decant off as much MMR as possible and add half of the dejelly solution. Swirl continuously for approximately 5 min. Dissolving jelly coats will be visible in the supernatant after a couple of minutes. Decant off and add the remaining dejelly solution. Continue to swirl continuously until eggs pack very tightly and all orient with their vegetal pole (the pole with white pigment) toward the bottom of the dish. Quickly decant off as much dejelly solution as possible. Once the eggs are dejellied they are very sensitive to mechanical manipulations.</li>
 <li>Carefully add XB to the eggs. In the first XB wash, remove eggs that have escaped CSF arrest by removing lysed, puffy, white, and pseudocleavage eggs. Activated X. tropicalis eggs tend to settle in the top center, so use a plastic transfer pipette to pull these out. Also remove pieces of skin and frog waste. Wash eggs a total of three times with ~ 300 ml 1X XB solution, gently swirling eggs between washes and allowing them to settle on the bottom of the beaker. As before, decant as much of each wash solution as possible while keeping eggs wet.</li>
 <li>Wash eggs twice with CSF-XB and decant.</li>
 <li>Add CSF-XB+ to eggs. Using a gelatin-treated fire-polished Pasteur pipette, transfer eggs to Ultra-centrifuge tubes with CSF-XB+, taking care not to expose the eggs to air. Place inside the 15 ml glass centrifuge tubes with the water cushion.</li>
 <li>Spin eggs in a clinical centrifuge at 200 x g for 1 min, increase speed to 800 x g and spin for 30 sec.</li>
 <li>Use an aspirator to remove as much buffer as possible from eggs. They should be almost dry on top. Quickly move eggs to a Sorvall RC-6 centrifuge equipped with a HB-6 rotor (or equivalent) and spin 17,000 x g for 15 min at 20 &deg;C.</li>
 <li>Remove the yellow cytoplasmic layer between the pigment and lipid layers using an 18 gauge needle attached to a 1 ml syringe. Puncture the side of the tube and pull the syringe barrel slowly to obtain the cytoplasmic extract layer. Avoid pigment granules as much as possible.</li>
 <li>Transfer cytoplasm to new ultracentrifugation tube. It is normal for the extract to appear slightly cloudy at this step. Place inside the 15 ml glass centrifuge tube with water cushion. Spin again 17,000 x g for 10 min at 20 &deg;C. Repeat extraction with 18 gauge needle.</li>
 <li>Transfer cytoplasm to a 1.5 ml microfuge tube. Estimate the extract volume and dilute Cytochalasin D and LPC 1:1,000 into the extract. Mix well with a 1 ml pipette tip, pipetting up and down many times without introduction of air bubbles. A typical yield from a healthy frog colony is approximately 300-500 &mu;l of extract/frog. To preserve maximum activity, it is necessary to store the extract and perform experimental manipulations at room temperature (20-25 &deg;C).</li>
</ol>
3. Purification Taxol-stabilized Microtubules from X. tropicalis Extract
<ol>
 <li>Add Taxol to a 100-200 &mu;l aliquot of extract at a final concentration of 10 &mu;M and incubate at room temperature for 30 min. For control reactions, treat an equivalent volume of extract with the microtubule-destablilzing drug Nocodazole (10 &mu;M). Reserve 100 &mu;l of untreated extract for analysis.</li>
 <li>Dilute the drug-treated extract with 10 volumes BRB-80 (80 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA) + 30% glycerol. Assemble 14 ml round-bottom polypropylene tubes containing 10 ml of BRB-80 + 60% glycerol cushion. Using a wide bore pipette tip, layer the drug-treated extract reaction gently on top of the BRB-80 + 60% glycerol cushion. Centrifuge for 10 min at 17,000 x g at 20 &deg;C in a Sorvall RC-6 centrifuge equipped with a HB-6 rotor (or equivalent) and tube adapters.</li>
 <li>Aspirate the supernatant containing unsedimented extract material, and wash the interface twice with deionized H2O. Aspirate the remaining cushion volume slowly, taking care not to disturb the gel-like pellet containing microtubules, microtubule-associated proteins, and microtubule-associated RNAs in the Taxol-treated sample. The Nocodazole-treated sample does not contain visible material. Resuspend the pellet in 1 ml TRIzol and proceed with the manufacturer's instructions for isolating RNA. Untreated extract (up to 100 &mu;l) can be resuspended directly in 1 ml TRIzol.</li>
 <li>There are now commercially available kits for preparing transcriptome libraries suitable for RNA-seq. These may be purchased through <a href="http://www.illumina.com/" target="_blank">http://www.illumina.com/</a> and <a href="http://www.454.com/" target="_blank">http://www.454.com/</a>.</li>
</ol>

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No conflicts of interest declared.

In this report we have described a simple method to produce CSF-arrested egg extracts from X. tropicalis19and use this extract to study microtubule-associated RNAs7. The basic procedure for producing CSF-arrested egg extracts from X. tropicalis is the same as used for X. laevis with a few key differences. One of the most challenging aspects to working with X. tropicalis frogs is obtaining enough high quality eggs to make an extract with microtubule nucleation or spindle assembly activity comparable to X. laevis egg extracts. To achieve optimal egg laying conditions while preventing slippage from meiosis II cell cycle arrest, the interval between hormone injections for X. tropicalis is shorter than that used for X. laevis, and the timing from the third hCG injection to the beginning of egg laying is also much shorter. With X. laevis the timing from the hCG injection to the beginning of egg laying is such that it is convenient and efficient for eggs to be laid overnight into buffer. However, because of the shorter time between hCG injection and egg laying with X. tropicalis it is frequently necessary to manually express the eggs from frogs. Another significant difference between making egg extract from the two different frogs is the dejellying step. With X. laevis the eggs are so large that it is easy to determine when the jelly coat has dissolved by observing how closely the eggs are spaced in the beaker. As the dejellying reaction commences, the eggs begin to pack more densely. However, X. tropicalis eggs are much smaller and it can be quite difficult to determine when the jelly coat has dissolved by egg packing density alone. We have found that the most reliable method to determine when the jelly coat has dissolved is to monitor the orientation of the animal (black) and vegetal (white) poles. When all the vegetal poles orient toward the bottom of the beaker the jelly coat has been removed enough to proceed with the extract. Finally, whereas X. laevis egg extract can be stored at cool temperatures (4-12 &deg;C) we have observed that it is critical to maintain X. tropicalis egg extract at room temperature (20-25 &deg;C) during preparation and experimental manipulations to preserve biochemical activity. Because of the differences in ease of use we prefer to use X. laevis frogs for the production of egg extract. However, for experiments that require or are facilitated by an organism with a sequenced genome, X. tropicalis is an excellent alternative system.
The method that we have described in this report uses taxol as a microtubule-stabilizing agent to induce microtubule polymerization. We chose this method because taxol is a robust microtubule-stabilizing agent that facilitates the large-scale isolation of purified microtubules. The method that we described could likely be improved by comparing the proteins and RNAs associated with microtubules using alternative microtubule polymerization methods. Alternatives could include polymerization using GTP-induced polymerization (a classic technique),22 or using Ran-GTP as a microtubule polymerizer to mimic the microtubules induced by chromatin-driven spindle assembly23. Finally, use of purified sperm nuclei to induce microtubule polymerization would be the closest mimic to the types of microtubules that are nucleated during mitosis (centrosome, chromatin, and kinetochore mediated). Drawbacks to these alternative sources of microtubule nucleation are that the nucleating agents are not as readily available as taxol and they do not nucleate or stabilize microtubules as efficiently as taxol. Therefore, each of these methods would be more difficult to use for large-scale purifications. The advantage of comparing multiple different types of microtubule nucleators is that it could be possible to identify proteins and/or RNAs that are specific to each pathway of microtubule nucleation.
The method that we have described here takes advantage of cytoplasmic extracts of amphibians. However, this approach could be extended to the use of extract system from other organisms. Mitotic extracts have been described from synchronized human tissue culture cells24 that faithfully recapitulate many aspects of microtubule assembly. We have successfully used these extracts to identify microtubule-associated RNAs from HeLa cells5. Similar microtubule purification schemes have been described for many different organisms25,26, although the microtubule associated RNAs have not been examined. The approach described here could be used with any organism that can produce a concentrated cytoplasmic extract capable of nucleating microtubules.
Finally, although the approach that we describe here discusses the purification of microtubules and associated proteins and RNAs, this approach could be generalized to other subcellular structures. While most localized mRNAs have not been identified using biochemical methods the recent advances in DNA and RNA sequencing technologies make this approach an attractive method to identify localized RNAs. In this approach any subcellular or sub-embryo structure of interest could be isolated or purified. Then the associated proteins and RNAs can be identified on a genome wide scale. RNAs can then be compared to the RNA content of the total cell or embryo to identify enriched localized RNAs. This approach could be used with whole eggs (animal and vegetal separation, similar to the approach that identified the first localized RNAs in Xenopus27), actin associated RNAs, ER-associated RNAs, mitochondria-associated RNAs, or to any subcellular structure that can be purified with associated RNAs intact. Based on our work on microtubule-associated RNA we predict that this would be an excellent method to discover new proteins that function at a given location. Furthermore, identification of the location and extent of all localized RNAs will provide insight into how cells and embryos use mRNA localization to control gene expression.

Spatial and temporal control of gene expression is important for all cells, and is especially important for the control of early embryonic pattering1. Spatial control of gene expression is achieved through the active localization of mRNAs to specific destinations within cells or embryos. In many very large cell types, (e.g. oocytes, embryos, and neurons) mRNA localization is used to restrict protein expression to the site of action of the coded protein. Since a localized mRNA can catalyze many rounds of protein production it is more efficient to localize an mRNA than to localize individual protein molecules. Localized mRNAs are typically translationally repressed until they reach their destination, which serves to further limit the localization of the coded protein2. In addition to the many well-documented cases of RNA localization to control embryonic patterning, several studies have documented mRNAs that are localized to the site of action of the encoded protein. Prominent examples include localization of the &beta;-actin3 and Arp2/34 mRNAs to the leading edge of motile fibroblasts and localization of the mRNAs for many mitotic regulators to meiotic and mitotic spindles5-7.
Many of the classic examples of localized mRNAs were identified through genetic screens for maternal effect mutations and were later determined to encode localized RNAs. However, recent genome-wide studies have begun to provide broader insight into the scope of localized RNAs. A recent in situ hybridization screen in Drosophila embryos demonstrated that ~70% of all mRNAs have a specific localization, including many novel destinations8. Purification of pseudopodia from mouse fibroblasts identified a diverse group of localized mRNAs9. Work from our group using biochemical purification of microtubules from meiotic Xenopus egg extracts identified hundreds of mRNAs that copurify with the spindle5,7. Our work showed that the majority of microtubule-localized mRNAs encode proteins that function in control of mitosis, supporting the idea that mRNAs are localized to the site of action of the coded protein. Furthermore, the ability to detect mRNA enrichment in a subcellular fraction by biochemical purification highlights the power of this approach for identification of localized mRNAs.
Most localized RNAs use active transport on the cytoskeleton, either actin or microtubules, to achieve transport to their final destination10. To gain a better understanding of the extent and types of RNAs that are localized to specific destinations using a biochemical approach it is necessary to have an in vitro system that can recapitulate cytoskeletal processes. One of the premier systems for studying cytoskeletal biology is egg extracts produced from unfertilized eggs from the frog Xenopus laevis. X. laevis egg extracts have been used for decades to study a wide array of cytoskeletal processes and have contributed much to our understanding of the mechanisms and molecules that control cytoskeletal assembly and dynamics11. Furthermore, X. laevis egg extracts are amenable to large-scale purifications of microtubules and associated proteins12,13 and there are well-designed methods for the production of various types of egg extracts14-16. However, for genomic studies there are several drawbacks to the use of X. laevis as a model system.
For decades Xenopus laevis frogs have been a powerful system for the study of developmental and cell biology, owing to the large oocyte size and robust external development17. Furthermore, the development of egg extract systems that can recapitulate many cellular processes in a test tube has made this frog a powerful experimental model. However, Xenopus laevis has been hampered by the lack of a complete genome sequence, which has been slowed by the allotetraploid nature of the genome.In contrast, a closely related species, Xenopus tropicalis, has a diploid genome that was sequenced in 201018. While X. tropicalis is not as experimentally tractable as X. laevis17the availability of a sequenced genome makes it an attractive model system to perform genome wide analyses.
In this report we describe a method to make meiosis II-, cytostatic factor-arrested extracts (CSF) from X. tropicalis19. We then describe a simple method to purify microtubules and associated RNAs from this extract. The RNAs can then be converted into libraries amenable to sequencing using recently developed high throughput sequencing technologies. Once the libraries are sequenced they can be aligned to the genome of the frog to identify specific mRNAs that are enriched in the microtubule sample compared to total extract. This provides a powerful method to detect microtubule-targeted mRNA localization on a genome-wide scale. In addition to being able to detect localized mRNAs, the use of high-throughput sequencing and a sequenced genome offer the possibility of discovering novel transcripts that are not currently present in public database annotations.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Xenopus tropicalis </td>
			<td>NASCO</td>
			<td>LM00823MX</td>
			<td></td>
		</tr>
		<tr>
			<td>human Chorionic Gonadotropin</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma-Aldrich</td>
			<td>L9783</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Sigma-Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr>
			<td>Chymostatin</td>
			<td>Sigma-Aldrich</td>
			<td>C7268</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin D</td>
			<td>Sigma-Aldrich</td>
			<td>C8273</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Taxol</td>
			<td>Sigma-Aldrich</td>
			<td>T7191</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine, free base</td>
			<td>USB Corporation</td>
			<td>14030</td>
			<td></td>
		</tr>
		<tr>
			<td>Cichlid Lake Salt</td>
			<td>Seachem</td>
			<td>47894</td>
			<td></td>
		</tr>
		<tr>
			<td>Marine salt</td>
			<td>Seachem</td>
			<td>SC7111</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>BD Biosciences</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge needles</td>
			<td>BD Biosciences</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gauge needles</td>
			<td>BD Biosciences</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubbermaid Plastic bucket</td>
			<td>Amazon</td>
			<td>6306</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Polyallomer 2 x &frac12; inch Ultracentrifuge tubes</td>
			<td>Beckman</td>
			<td>326819</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml round-bottomed glass centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>45500-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber adapter sleeves for 15 ml tubes</td>
			<td>Kimble-Chase</td>
			<td>45550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>5 &frac34; inch glass Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml polypropylene round-bottom tube</td>
			<td>BD Biosciences</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall HB-6 rotor</td>
			<td>Thermo Scientific</td>
			<td>11860</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall RC-6 centrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of xenopus tropicalis egg extracts to identify microtubule-associated rnas

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

To identify X. tropicalis transcripts associated with microtubules, we prepare a cytosolic extract from unfertilized eggs arrested in metaphase of meiosis II (CSF). Treatment of this extract with taxol allows the formation of stable microtubules that can be purified by sedimentation through a glycerol cushion (Figure 1A). Coomassie gel analysis confirms that &alpha;/&beta;-tubulin sediments in a taxol-dependent manner, and represents the major protein species recovered in these preparations (Figure 1B). Lower levels of other proteins are also present in the taxol pellet, but not in preparations treated with the microtubule depolymerizing drug nocodazole, indicating that proteins in the taxol fraction specifically associate with microtubules (MAPs).
An Agilent Bioanalyzer is used to examine general RNA composition in all X. tropicalis extract fractions (Figure 1C). Both rRNA and tRNA species are present in CSF extract and the microtubule-containing taxol pellet, consistent with previous findings that translation occurs on microtubules and spindles in X. laevis egg extract5,21. A line trace of the gel projection reveals the mRNA signal is markedly lower in the microtubule-containing taxol pellet, most notably in the region migrating above 28S rRNA, indicating that a subset of mRNAs cosediment with microtubules in X. tropicalis. RNA isolated in this manner is suitable for RNA-seq experiments using commercially available reagents.
<img src="/files/ftp_upload/50434/50434fig1.jpg" alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50434/50434fig1hires.jpg" /> 
Figure 1. Purification of MT-RNA for RNA-seq. (A) Purification scheme to isolate MT-RNA. Eggs are harvested from female X. tropicalis frogs. After preparation of a cytoplasmic extract, taxol is added to induce microtubule polymerization. Microtubules and MT-RNA are purified by sedimentation through a glycerol cushion. (B) Coomassie gel analysis of proteins isolated using the scheme described in (A). Total CSF extract compared to proteins sedimented in the presence of taxol or nocodazole. (C) Bioanalyzer gel analysis of RNA isolated using the scheme described in (A). RNA isolated from CSF extract compared to RNA sedimented in the presence of taxol or nocodazole. Both the gel projection and the line traces are shown. Reprinted with permission from Sharp, et al., (2011). <a href="https://www.jove.com/files/ftp_upload/50434/50434fig1hires.jpg">Click here to view larger image</a>.

Watch this Scientific Journal Video about Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs at JoVE.com

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

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

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

production-xenopus-tropicalis-egg-extracts-to-identify-microtubule

Research

JoVE Journal

Biology

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

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 ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...