A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

The overall goal of this experiment is to faithfully reconstitute chromatin de condensation as it happens at the end of mitosis in the simplicity of a cell-free assay. This method can help answer key questions in the fields of mitosis research as it enables the characterization of the molecular mechanism of chromatin, decolonization, and the identification of the factor involved in the regulation. The main advantage of this technique is that it can be easily biochemically manipulated and chromatin.

Decolonization can be studied in isolation separated from other cell cycle events. To begin isolate mitotic chromatin clusters from helo cells and prepare the buffers for the preparation of IIC xenopus lavis egg extract as described in the accompanying text protocol. Once prepared, use a five milliliter syringe equipped with a 27 gauge needle to inject 500 international units of human chorionic gonadotropin into ovulation induced frogs.

Inject the frogs under the skin of the dorsal lymph sac on the evening before the experiment to induce the release of the eggs Once induced, keep the frogs at 18 degrees Celsius for 13 to 17 hours in individual tanks containing 1.2 liters of mark's modified ringers buffer. After the frogs lay their eggs, remove the eggs that are already activated along with any bad looking eggs. Collect them by pouring out the tanks into 600 to 1000 milliliter glass beakers Correctly.

Sorting the eggs is critical as the overall extra quality depends on the step. Remove activated stringy and otherwise bad looking eggs. Also in later steps, continue to remove bad eggs.

Decant the S supernatant once the eggs settle, and then refill the beaker with fresh buffer afterwards. Repeat this process four times to wash the eggs following the last wash. Remove the supinate one more time and then fill the beaker with a 2%L cysteine solution to delineate the eggs.

After two to four minutes, change to fresh buffer containing L cysteine. Consider de geling complete when the volume of the eggs drastically decreases and the eggs become more densely packed after approximately five to seven minutes. Next, carefully wash the eggs in mark's modified ringers buffer by decanting and refilling the buffer supinate on the wall of the beaker instead of directly onto the eggs.

Then activate the eggs in 100 milliliters of Marx modified ringers buffer containing eight microliters of two milligrams per milliliter. Calcium ion. Four, stop the activation when the animal cap contraction becomes visible.

Or after 10 minutes following activation, wash the eggs carefully in mark's modified ringer's, buffer by decanting and refilling the buffer supinate after the last wash. Incubate the eggs in room temperature for 20 minutes in mark's modified ringer's buffer. Next, prepare five milliliters centrifugation tubes with 50 microliters of sucrose buffer, 50 microliters of 100 fold protease inhibitor.

Mix five microliters of one molar DTT 12.5 microliters of cyclo heide, and 2.5 microliters of cyto. B.Wash the eggs twice with cold sucrose buffer, and then transfer them into the centrifugation tubes. Using a plastic pasture pipette with a wide opening pack eggs by spinning the tubes for one minute at 130 G.After centrifugation, remove the excess buffer using a plastic pasture pipette and fill in the extra room with more eggs.

Then centrifuge the egg filled tubes in a six by five milliliter swing rotor for 20 minutes at four degrees Celsius and 21, 000 times g. With the solution now separated into three layers, push a five milliliter syringe with a 16 gauge needle through the side of the tube and into the middle layer between the yellow yolk on the top and just above the dark broken egg debris on the bottom. Transfer the low speed phasic extract into a fresh tube on ice, and then add 10 microliters of a 100 fold protease inhibitor.

Mix one microliter of one molar DTT 2.5 microliters of cyclo heide at 20 milligrams per milliliter, and 0.5 microliters of cyto B.Add 10 milligrams per milliliter to each milliliter of the low speed extract. Spin the low speed phasic extract for 12 minutes at 355, 000 times G in a 10 by two milliliter rotor. Then gently remove the lipid layer on top and transfer the supinate containing the high speed extract into a fresh tube.

This step is critical for producing a high quality sample. Take care that the liquid removed does not contain contaminations from the lipid layer on top or the annoying bottom layer. Pipette 18 microliters of the high speed extract into a 1.5 milliliter reaction tube, and then add 0.5 microliters of glycogen 0.5 microliters of the energy mix.

Point three, microliters of six dimethyl purine, and 0.7 microliters of the mitotic clusters. Use tips with a wide opening to mix the reaction as soon as the chromatin is added in order to prevent shearing of the decompensating chromatin. Once mixed, incubate the reaction mixture for up to two hours at 20 degrees Celsius.

Then fix the sample by adding 0.5 milliliters of ice cold fixative containing 4%paraldehyde 0.5%glutaraldehyde and 0.1 milligram per milliliter dpi. Using tips with a wide opening, incubate the sample on ice for 20 to 30 minutes. Next, code 12 millimeter round cover slips with a 0.1%weight to volume poly L lysine solution and incubate the cover slips for five minutes to increase the affinity of the cover slips.

To chromatin dry the cover slips on filter paper afterwards. Once dry, add the coated cover slips to the bottom of six milliliter flat bottom centrifugation tubes so that the coated side is up. Then add 800 microliters of the 30%sucrose cushion and layer the entire fixed sample on top.

Spin the sample for 15 minutes at four degrees Celsius and 2, 500 times G.Then decamp the S supernatant and remove the cover slips from the tubes by carefully poking at the bottom of the centrifugation tube with a 16 gauge needle. When the cover slip is lifted by the needle on one side, use tweezers to remove the cover slip, wash the cover slip quickly by dipping it in deionized water, and then dry it gently by touching its side to filter paper. Next, place a drop of mounting media onto a microscope slide and lower the sample side of the cover.

Slip onto the mounting media. Dry the cover slip gently with a paper wipe seal around the edge of the cover slip with nail polish and keep the slide in the dark until it is imaged. Here is the typical time course of the decompensation assay.

The cluster of chromosomes visible at the beginning of the reaction, decon, condenses and merges into a single round and smooth nucleus. When the egg extract is replaced by sucrose buffer, the chromosome cluster remains condensed, which suggests that decompensation activity is present in the egg extract. In the experiment shown here, non hydrolyzed A TP or GTP analogs were added to the reaction.

Both of these additives inhibit the de condensation proving that this active process is both A TP and GTP hydrolysis dependent. Once mastered, this technique can be done in approximately two hours for the Singapore levels egg extract preparation, and three hours for the promoting de colonization assay. While attempting this procedure, it's important to remember that the success critically depends on the extract quality.

Especially for immuno depletions, it is recommended to use freshly prepared extract following this procedure. Other methods like immuno depletions and the addition of recombinant proteins can be performed in order to answer additional questions like which factors are involved. This technique will pave the way for researchers in the fields of mitosis or nuclear structure and function to explore chromatin dehumanization as it happens in the end of mitosis in animal cells.

After watching this video, you should have a good understanding of how to prepare ex xenopus liver egg extracts and how to apply the method to reconstitute chromatin decolonization. Don't forget that working with Paraform Hyde, Hyde and DARPA can be hazardous, and precautions should be taken, such as wearing gloves.