The overall goal of the following experiment is to produce a cytoplasmic extract from unfertilized xenopus tropic callus eggs that can be used to recapitulate many cellular processes in vitro. This is achieved by first injecting hormones to induce ena post tropic callus to lay unfertilized eggs. Next, the eggs are collected, washed and crushed by centrifugation and the egg cytoplasm is isolated.
Then the extract is used to purify microtubules associated proteins and RNAs to gain a genome-wide view of mRNA. Localization results are obtained that show hundreds of mRNAs specifically associate with myotic microtubules based on high throughput sequencing of microtubule associated RNAs. The main advantage of this technique over existing methods like Opus Lavis egg extracts is that zap tropic have a sequence genome which allows comprehensive identification of transcribed sequences and express proteins, which is not currently possible with zap lavis To generate X tropic eggs for isolating extracts.
Begin by preparing two concentrations of human chorionic gonadotropin or HCG. First, we suspend 10, 000 units of Lyophilized HCG powder in 10 milliliters of sterile deionized water for a final concentration of 1000 units per milliliter. Then combine one milliliter of the diluted solution with nine milliliters of water for a final concentration of 100 units per milliliter.
Store both solutions at four degrees Celsius on day one at about two to 3:00 PM prepare four to six frogs for egg laying by injecting them in the dorsal lymph sac near the cloaca with 0.2 milliliters of 100 units per milliliter of HCG to minimize the amount of frog waste present during the subsequent injections. Have the frogs fast on day two at two to 3:00 PM Administer a second identical injection to the same frogs the following day. Repeat the injection between seven and 10:00 AM as indicated here to set up the frogs for egg laying, fill a six quart plastic bucket with fresh tank water.
Add the frogs and place it in the dark at 25 degrees Celsius. Egg laying should begin within four hours and be completed by seven hours. In the meantime, prepare buffers solutions and drugs according to the text protocol.
Next to make fire polished glass pastier pipettes, snap the end off a five and three quarter inch glass pipettes to make a broad opening and expose them to a flame. To smooth the tip. Prepare a 500 milliliter glass beaker for storing eggs by swirling a 0.2%gelatin solution around to coat the walls of the beaker.
Then discard the unused gelatin solution. When egg laying is complete, collect the eggs from the plastic bucket and if desired, gently squeeze each frog once to isolate any remaining eggs. Use tank water to wash the eggs once and transfer them to the gelatin coated glass beak with just enough tank water to keep the eggs wet.
To prepare extract slowly, add about 300 milliliters of one XMMR to the wall of the beaker to minimize physical agitation to the eggs, let them settle. Decant the supernatant to remove any debris and repeat the MMR wash. Two more times next to dey the eggs decant off as much MMR as possible and add 125 milliliters of freshly prepared dey solution Swirl continuously for about five minutes.
After a couple of minutes, the dissolving jelly coats will be visible. At this point, decant the liquid and add the remaining 125 milliliters of fresh de jelly solution. Again, continuously swirl the solution until the eggs pack very tightly and they all orient with their vegetal poles towards the bottom of the dish.
The now de jelly eggs are sensitive to mechanical manipulation. Quickly decant off as much liquid as possible and carefully add one X XB wash solution and swirl, allowing the X to settle to the bottom of the beaker activated. X tropicalis eggs that have escaped cytostatic factor or CSF arrest will appear laced.
Puffy white and as having undergone pseudo cleavage tend to remain at the surface. Use a plastic transfer pipette to remove these along with any pieces of skin and frog waste and decant as much solution as possible before repeating the wash. Two more times, then wash the eggs twice with CSF XB and decant before suspending in CSF xb.
Plus, using a gelatin treated fire polished past pipette. Transfer the eggs to ultracentrifuge tubes with CSF XB plus taking care not to expose the X to air. Place the tubes inside 15 milliliter glass centrifuge tubes containing a water cushion and spin the eggs in a clinical centrifuge at 200 Gs for one minute.
Increase the speed to 800 GS and spin for 30 seconds. Use an aspirator to remove as much buffer as possible from the eggs. They should be almost dry on top.
Then quickly transfer the eggs to a sova RC six centrifuge equipped with an HB six rotor and spin at 17, 000 GS and 20 degrees Celsius for 15 minutes. With an 18 gauge needle attached to a one milliliter syringe. Remove the yellow cytoplasmic layer between the pigment and lipid layers.
Then puncture the side of the tube and pull the syringe barrel slowly to collect the cytoplasmic extract layer, avoiding the pigment granules as much as possible. Transfer it to a new ultracentrifugation tube. Place into a glass centrifuge tube with a water cushion and spin at 17, 000 GS at 20 degrees Celsius for 20 minutes.
After repeating the extraction a second time, transfer the cytoplasm to a 1.5 milliliter micro fuge tube and estimate the volume, which is typically 300 to 500 microliters of extract per frog from a healthy frog colony. Then add cyto D and leptin PEPs statin and chios statin or LPC. Add final concentrations of one to 1000 each.
Use a one milliliter pipette tip to mix well, avoiding air bubbles to preserve maximum activity. Store the extract and perform experimental manipulations at room temperature to a 100 to 200 microliter aqua extract. Add taxol at a final concentration of 10 micromolar and incubate it at room temperature for 30 minutes.
For control reactions, treat an equivalent volume of extract with the microtubule destabilizing drug, no conazole and reserve 100 microliters of untreated extract for analysis, dilute the drug treated extracts with 10 volumes of BRB 80 plus 30%glycerol, then assemble 14 milliliter round bottom polypropylene tubes containing a 10 milliliter cushion of B rrb 80 plus 60%glycerol. Next, using a wide bore pipette tip, gently layer the drug treated extract reaction on top of the BRB 80 plus 60%glycerol cushion and use tube adapters to centrifuge at 17, 000 GS and 20 degrees Celsius for 10 minutes. Aspirate the supernatant containing un sedimented extract material and wash the interface twice with deionized water for the Taxol treated sample.
Slowly aspirate the remaining cushion volume. Taking care not to disturb the gel-like pellet containing microtubules, microtubule associated proteins and microtubule associated RNAs. The no cortisol treated sample will not contain visible material.
We suspend the pellet in one milliliter of triazole and proceed with the manufacturer's instructions for isolating RNA up to 100 microliters of untreated extract can be resuspended directly in one milliliter of triol. Finally, use a commercially available kit for preparing transcriptome library suitable for RNA sequencing In this figure. Kumasi gel analysis of microtubules purified from X tropic extracts treated with Taxol, confirms that alpha beta tubulin sediments in a Taxol dependent manner and represents the major protein species recovered in these preparations.
Lower levels of additional proteins are also present in the Taxol pellet, but not in preparations treated with the microtubule depolymerizing drug nool indicating that proteins in the Taxol fraction are micro tubule associated proteins as seen here. Analysis of RNA composition of X tropic extracts indicates that both RRNA and TRNA are present in CSF extracts and the microtubule containing Taxol pellet consistent with previous findings. That translation occurs on microtubules and spindles in X lavis egg extracts.
A line trace of the gel projection shown here reveals the mRNA signal is markedly lower in the micro tubule containing Taxol pellet, most notably in the region migrating above 28 S-R-R-N-A, indicating that a subset of mRNAs co sediment with microtubules in x tropic Following this procedure. Other methods like cell cycle analysis can be performed in order to answer additional questions such as how localized mRNAs affect myotic spindle assembly.
