Ein zellfreien Assay unter Verwendung von

The overall goal of this procedure is to use Xenopus egg and embryo extracts to produce an assay in which large nuclei shrink smaller. This method can help answer key questions about nuclear cell biology, such as what are the mechanisms that regulate nuclear size? The main advantage of this technique is that the composition and activities of Xenopus cell-free extract are easily manipulated to assess the facts on nuclear size.

After preparing Xenopus egg extracts, demembranated sperm, and assembled nuclei according to the text protocol, collect freshly laid eggs by holding frogs over a glass, reusable Petri dish. Promote egg-laying by applying gentle pressure to the lower back near the cloaca. Prop up the dish at approximately 45 degrees to allow the eggs to collect along the edge of the dish.

Then, remove the excess buffer and add enough one-third X MMR to barely cover the eggs. Using a tightly-fitted pestle, macerate and homogenize 1/4 of a testis in a 1.5 milliliter tube with 500 microliters of high-salt Modified Barth's Saline, or MBS. Then add the macerated testis to the eggs and allow fertilization to occur at room temperature for 15 minutes.

After the incubation, flood the eggs with one-third X MMR. Then, five to 10 minutes later, confirm fertilization by checking for contraction of the dark pigmented animal pole and by rotation of the embryos with their animal poles facing up. Using a wide bore plastic pipette, diligently remove dead, lysed, or unfertilized, uncleaved eggs as they will rapidly induce death in neighboring embryos.

Anywhere from one to 2.5 hours post-fertilization, dejelly embryos in two to three percent cysteine dissolved in one-third X MMR, which has been adjusted to pH 7.9. Pryor to dejellying, embryos occupy a relatively large volume. Perform two buffer changes for three to four minutes each.

Then, thoroughly wash the embryos with six to 10 brief changes of one-third X MMR to remove all traces of cysteine. After dejellying, embryos pack tightly and occupy a relatively small volume. Next, using a wide-bore glass pipette, transfer healthy, fertilized embryos to a Petri dish containing fresh one-third X MMR.

And at room temperature, allow them to develop to the desired stage. Continue to remove dead or lysed embryos as indicated by a lack of first division or a white, puffy appearance. When the embryos have reached stage 11.5 to 12, transfer them into fresh one-third X MMR supplemented with 0.5 millimolar cycloheximide and incubate the embryos at room temperature for one hour to arrest them in late interphase.

With a wide bore glass or plastic pipette, transfer a minimum of 15 interphase arrested embryos to a microcentrifuge tube. Add one millileter of egg lysis buffer, or ELB, supplemented with one microliter of LPC stock to the embryos. Gently invert the tube to wash the embryos.

Then allow the embryos to fall to the bottom of the tube. Before removing the buffer, repeat the wash two more times. Resuspend the embryos in 500 microliters of ELB, plus 0.5 microliters of LPC stock, 5 microliters of 19.7 millimolar cycloheximide, and 5 microliters of 35.5 millimolar cytochalasin D to inhibit actin polymerization.

Centrifuge the samples in a tabletop centrifuge at 200 times G for one minute, then remove the excess buffer and use a pestle to thoroughly crush the embryos. Place the crushed samples in a swinging bucket rotor and centrifuge at 10, 000 times G, and 16 degrees Celsius for 10 minutes. With a 200 microliter pipette tip, puncture the lipid layer from the top, and using a clean pipette tip, remove the middle cytoplasmic amber-colored layer to an appropriately-sized tube.

Supplement the embryo extract with the following reagents. Then, gently invert the tube to mix. Then, after visualizing endogenous embryonic nuclei in the extract by preparing a squash according to the text protocol, remove the nuclei from the extract by first adding an equal volume of ELB containing the reagents listed here.

Gently invert the tube to mix. Centrifuge the sample in a swinging bucket rotor at 17, 000 times G at 16 degrees Celsius for 15 minutes. Then, collect the supernatant, taking care to avoid any remaining lipid at the top, and leave the pelleted nuclei at the bottom undisturbed.

Prepare a squash as described in the text protocol to ensure that most nuclei have been removed. Then use the fresh extract, or aliquot the sample, and store it at minus 80 degrees Celsius. Isolate nuclei assembled in egg extract by diluting 25 to 150 microliters of pre-assembled nuclei in one millileter of ELB in a 1.5 milliliter tube.

Centrifuge at 1600 times G and four degrees Celsius for three minutes. Then remove the buffer, taking care not to disturb the pellet. Add to the pellet a volume of embryo extract equal to the original volume of egg extract, and gently tap the tube to break up the pellet and resuspend the nuclei.

Incubate at room temperature for 90 minutes, gently tapping the tube to mix every 15 to 30 minutes. After the incubation, tap the tube to resuspend the nuclei just prior to adding 500 microliters of ELB with 15%glycerol and 2.6%Paraformaldehyde. Invert the tube immediately, and place the tube on a rotator at room temperature for 15 minutes.

Prepare spin-down tubes by outfitting 15 milliliter, round-bottom glass tubes with round-bottom plastic inserts. Add 5 milliliters of nuclear cushion buffer, then drop a 12 millimeter circular coverslip into the tube, being sure it lays flat on top of the plastic insert. Using a wide bore pipette tip, gently layer the fixed nuclei solution on top of the nuclear cushion.

Centrifuge it in a swinging bucket rotor at 1, 000 times G and 16 degrees Celsius for 15 minutes. Next, use an aspirator to remove all the buffer and hold the plastic insert to the top of the tube. Then, with a pair of fine forceps, remove the coverslip, taking care to note the side of the coverslip onto which the nuclei were spun.

Host fix the coverslip in cold methanol for five minutes at room temperature. Transfer the coverslip, nuclei-side up onto a sheet of plastic paraffin film lining a large, plastic Petri dish. Then place wet, disposable wipes along the side of the dish to prevent dehydration.

With 500 microliters of PBS NP40, rehydrate the nuclei on the coverslip for five to 10 seconds and aspirate. Then, carefully layer 75 microliters of PBS 3%BSA onto the coverslip, and allow it to block at room temperature for one hour, or four degrees Celsius overnight. Remove the blocking buffer and incubate with primary antibody diluted in PBS 3%BSA.

Then wash the samples with five immediate changes of PBS NP40. Repeat these incubation and washing steps with secondary antibodies. Incubate with 10 micrograms per milliliter of Hoechst dilated in PBS 3%BSA for five minutes at room temperature.

Then wash five times with PBS NP40 and remove all excess buffer. Finally, mount the coverslip onto a slide with five microliters of anti-fade mounting medium. Use clear nail polish to seal the coverslip and immediately image using an epifluorescence microscope or store at 40 degrees Celsius for later imaging.

This figure shows the assembly of egg extract nuclei. 30-45 minutes after initiation of the reaction, the thin, S-shaped sperm nuclei should begin to thicken into slug-shaped chromatin masses. After nuclear assembly occurs, the nuclear shape should become more rounded, and the nuclear volume should increase as the nuclear envelope expands and nuclear proteins are imported.

As shown here, a Hoechst stained squash of a small aliquot of the embryo extract shows many stained nuclei indicating that the preparation of extract was successful. After nuclei removal by centrifugation, compared to the first squash, very few nuclei should be left in the extract, as revealed in this panel, ensuring that endogenous nuclei do not interfere with downstream analysis. If nuclei are still present, a second round of centrifugation is required.

In this nuclear shrinking assay, egg extract nuclei resuspended in late-stage embryo extract, successfully becomes smaller over time. However, nuclei incubated with heat inactivated embryo extract do not. Repeating the experiment multiple times is important to address inherent variability of the assay.

Once mastered, the entire protocol can be done in two days with the nuclear shrinking assay itself taking approximately four hours. While attempting this procedure, it's important to remember to quality-check the eggs and embryos throughout and to be very careful to avoid the pellet and lipid layers when collecting cytoplasmic extract after centrifugation. Results obtained by following this procedure should be validated.

For example, my embryo microinjection, or cell culture transfection. Furthermore, this method might be used to explore size regulation of other organelles. After its development, this technique paved the way for researchers in the field of nuclear cell biology to explore the regulatory mechanisms of nuclear size and the functional role of nuclear size in development and carcinogenesis.

After watching this video, you should have a good understanding of how to assemble and isolate Xenopus egg extract nuclei, generate Xenopus embryos and embryo extract, perform the nuclear shrinking assay and visualize the results.