The overall goal of the following experiment is to assay gene expression using functional nuclear machineries extracted from cells. This is achieved by first harvesting in swelling the hela cells. Next, the cells are laced by downing and then centrifuge to pellet the nuclei in the third step.
The nuclei are salt extracted, concentrated, and dialyzed in order to obtain soluble and functional gene expression machineries. Ultimately, the functionality of the nuclear extracts can be evaluated based on their transcription and splicing activities as assessed with a coupled RNA polymerase to transcription and splicing system. The main advantage of this technique over existing methods like bulk nucle extract preparation, is that with this technique, a small amount of cells can be used to prepare the nuclei extract.
Begin by plating hela cells on three 150 millimeter plates with DMEM media supplemented with FBS and antibiotics grow the cultures in a 37 degree Celsius incubator with 5%CO2 until the cells reach 90%Co fluency at this point aspirate the media from the HELOC cell cultures and wash them once with 13 milliliters of room temperature. PBS per plate then aspirate. The PBS stand each plate on its side for a few seconds and aspirate the rest of the PBS.
Next, use a cell lifter to scrape the cells to the bottom edge of each plate, and then pull the cells in a 1.5 milliliter einor tube. Finally, pellet the cells in a micro fuge for five minutes at a hundred times G and at four degrees Celsius. Now aspirate the PBS from the cell pellet and estimate the packed cell volume or PCV by looking at the gradation on the tube.
Then add twice the volume of hypotonic buffer as the PCV to the cell pellet. Shake the tube gently to resuspend. The cells gently pipetting the cell suspension up and down with a 1000 microliter pipette to dislodge cell clusters as necessary.
Then pellet the cells under the same centrifuge conditions as before. After centrifugation carefully aspirate the hypotonic buffer layer. The cells will have begun to swell and the cell volume will have increased to about 750 to 1000 microliters.
Add new hypotonic buffer to a final volume of 1.5 milliliters and resuspend the cells in the buffer.Again. Finally, incubate the resuspended swollen cells on ice for 10 minutes while the cells are chilling. Rinse a down homogenizer with hypotonic buffer and then place it on ice along with the cells.
Then using a delicate task wiper such as a Kim wipe, dry the pestle. Use a 1000 microliter extended micro pipette tip to remove the buffer from the downs. Now use the pipette to transfer the chilled swollen cells into the downs Down.
Is the trickiest part of this procedure to an enter success Check for lysis. After each downs at the microscope, Slowly move the tight pestle of the downs up and down five times to lice the cells being careful to avoid bubbles. Next, transfer a five microliter Eloqua of the lys cell suspension to an einor tube and add 10 microliters of trian blue and 15 microliters of one XPBS.
Add the cell solution to a slide and examine the cells under a light microscope for lysis, the nuclei of lys cells will appear blue after three ounces. There is not enough lysis as numerous living cells are visible. After five ounces, there is enough lysis.
Finally transfer the lys cells to the pre chilled einor tube and spin down the cell suspension for five minutes in a micro fuge at 1500 times G and four degrees Celsius. Then carefully transfer the super natin to a new tube without disrupting the pellet. Now, estimate the patch nuclear volume or PNV by looking at the gradation on the tube.
Fill a micro pipette tip with low salt buffer at one half the PNV. Then insert the micro pipette tip into the nuclei at the bottom of the einor tube. Slowly squirting the buffer into the nuclei while gently mixing.
Gently flick the tube to make sure that the pellet is completely resuspended. Next, add high salt buffer at one half the PNV and quickly mix one time by inverting the tube. Rotate the tube for 30 minutes at four degrees Celsius.
Finally, spin down the nuclei in the micro fuge for 15 minutes at 18, 000 times G and four degrees Celsius. Begin by transferring 500 microliters of the supernatant into chilled micro cons. Spin down the high salt nuclear extract for 15 minutes at 14, 000 times G and four degrees Celsius after centrifugation, invert the mini racon and place it into a new individual EOR tube.
After spinning for two minutes at 1000 times G and four degrees Celsius, the high salt nuclear extract will be concentrated to approximately 115 microliters. Now transfer 45 microliter aliquots of the high salt nuclear extract into mini dialysis SLIs. Ensure that the bottom of the slide are with the bottom of the floater.
Then place the SLIs in 500 milliliters of chilled dialysis buffer. Finally, stir the aliquots for one to two hours at four degrees Celsius, the nuclear extract will appear cloudy after dialysis. This figure shows representative data comparing a coupled RNA polymerase two transcription and slicing reaction in a small scale nuclear extract versus a bulk nuclear extract.
When A-C-M-V-D-N-A construct was incubated in the bulk or small scale extracts, similar levels of the nascent pre mRNA were synthesized by the five minute time point following addition of alpha and manin to block further transcription, the splicing intermediates and splice products accumulated over time with similar kinetics in both types of extracts. These representative results confirm that the efficiencies of the coupled RNA polymerase two transcription and splicing system are similar in the bulk and small scale nuclear extracts. To demonstrate the utility of the small scale extract, method extracts were prepared from hela cells treated with the splicing inhibitor E 7 1 0 7 or the negative control compound Platy analy F and then a coupled RNA polymerase two transcription and splicing assay was carried out as demonstrated in this figure transcription by RNA polymerase two occurred efficiently in extracts prepared from both the plaid F and E 7 1 0 7 treated cells.
In contrast, splicing occurred normally in the extract prepared from the analy F treated cells, but was abolished in the E 7 1 0 7 treated cells. These data provide proof of concept for using the small scale nuclear extracts for special treatment of cells in small scale. Once mastered, this technique can be completed in less than five hours.
