Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

1. Growing HeLa Cells for Nuclear Extraction

1. Plate HeLa cells on three 150 mm plates with DMEM media supplemented with 10% FBS and 1% Penicillin/Streptomycin. Grow in a 37 °C incubator with 5% CO₂ until cells are 90% confluent.

Tip- Split cells 1:10 from a confluent plate and harvest 3 days after splitting.

Tip- An extra plate can be grown in case there are not enough cells with three plates (for example, if the cells are less than 90% confluent).

2. Prepare and Chill Nuclear Extract Solutions

1. Prepare fresh solutions as outlined in Table 2.
2. Aliquot 10 ml of Hypotonic Buffer into a 15 ml Falcon tube. Aliquot 1 ml of High Salt Buffer and 1 ml of Low Salt Buffer into 1.5 ml Eppendorf tubes. Chill the buffers on ice. Add the appropriate volumes of PMSF and DTT to each buffer immediately prior to use (see Table 1).

3. Harvesting HeLa Cells for Nuclear Extraction

1. Aspirate media from the cells and wash the cells once with 13 ml of room temperature 1X PBS per plate.
2. Aspirate the PBS wash.

Tip- Aspirate as much of the PBS as possible by first aspirating, then standing the plate on its side, waiting a few seconds, and aspirating the rest.

3. Using a cell lifter, scrape cells to the bottom edge of the plates, and then transfer the cells to an Eppendorf tube. Avoid making bubbles. The 3 plates of scraped cells should fit into one 1.5 ml Eppendorf tube.

Tip- It is easier to estimate the volume of cells if an Eppendorf tube is used. For scale up, use Falcon tubes.

4. Centrifuge at 4 °C in a microfuge for 5 min at 100 x g to pellet the cells.

4. Swell HeLa Cells in Hypotonic Buffer

1. Carefully aspirate the PBS from the cell pellet and estimate the Packed Cell Volume (PCV) by looking at the gradation on the tube. Three confluent plates of HeLa cells yield about 500 μl of PCV.
2. Add 2X the PCV of Hypotonic Buffer to the cell pellet. Shake gently to resuspend the cells. Use a P1000 Pipetman to gently resuspend the cells by pipetting up and down if necessary.
3. Centrifuge at 4 °C in a microfuge for 5 min at 100 x g to pellet the cells. *The cells will have begun to swell by this time, and the cell volume should now be about 750 μl-1000 μl.
4. Carefully aspirate the Hypotonic Buffer and add new Hypotonic Buffer to a final volume of 1.5 ml. Resuspend the cells.
5. Incubate the resuspended swollen cells on ice for 10 min.

5. Lyse Cells Using a Dounce Homogenizer

1. Use Hypotonic Buffer to rinse the dounce and then chill it on ice. Wipe the pestle dry with a delicate task wiper (e.g. Kimwipe). Remove the remaining buffer from the dounce using a 1000 μl extended micropipette tip.
2. Transfer swollen cells into the dounce. Slowly move the tight pestle of the dounce up and down 5 times to lyse the cells, being careful to avoid bubbles.
3. Assay for cell lysis using a 5 μl aliquot of cells from the dounce. Place the aliquot in an Eppendorf tube and add an equal volume of Trypan Blue. Put the mixture on a slide and examine the cells using a light microscope. The nuclei of lysed cells will appear blue. About 90% lysis is ideal.

Tip- The number of times the cells should be dounced will vary depending upon the tightness of the dounce. When carrying out this protocol for the first time, determine the number of times to dounce by carrying out step 5.3 after each stroke with the dounce. Do not over-dounce, excessive douncing will destroy the nuclei.

4. Transfer the lysed cells to a pre-chilled Eppendorf tube and spin in a 4 °C microfuge for 5 min at 1500 x g. The pellet contains the nuclei. Carefully transfer the supernatant to a new tube without disrupting the nuclei. The supernatant contains the cytoplasm.

Tip- The cytoplasm can be used as is or further processed to an S100 by using the same high-speed spin used for bulk extracts (see¹ for preparing an active S100 from bulk extracts).

6. Salt-Extract the Nuclei

1. Estimate the Packed Nuclear Volume (PNV) by looking at the gradation on the tube. A PCV of 500 μl typically yields a PNV of 400 μl.
2. Add ½ X PNV of Low Salt Buffer to the nuclei by inserting a micropipette tip to the bottom of the tube and slowly squirting the buffer into the nuclei while gently mixing. Do not pipet up and down. Gently flick the tubes to make sure that the pellet is completely resuspended in the Low Salt Buffer before continuing.
3. Add ½ X PNV of High Salt Buffer and quickly mix 1 time by inverting the tube. Do not shake. Rotate at 4 °C for 30 min.

Tip- Be gentle with the nuclei when they are in High Salt Buffer to avoid lysing them.

4. Spin at 4 °C in a microfuge for 15 min at 18,000 x g. The supernatant is the high salt nuclear extract (HS-NE). Three plates of cells typically yield 500-600 μl of HS-NE.

7. Concentrate and Dialyze the Nuclear Extract

1. Transfer 500 μl of the HS-NE into chilled mini-centricons (Amicon) and spin for 50 min at 4 °C in a microfuge at 14000 x g. Invert the mini-centricon (Amicon) and place it in a new Eppendorf tube. Spin for 2 min at 4 °C 1000 x g to recover the HS-NE. The HS-NE will be concentrated to approximately 115 μl.
2. Using a P200 Pipetman, transfer 45 μl aliquots of the HS-NE into mini-dialysis Slide-A-Lyzers, and place them in 500 ml of chilled Dialysis Buffer. Ensure that the bottom of the Slide-A-Lyzer is aligned with the bottom of the floater. Stir for 1-2 hrs at 4 °C. The NE will appear cloudy after dialysis.

Tip- Do not centrifuge to remove the cloudy precipitate that is observed after dialysis, as this centrifugation reduces the activity of the extract.

3. The NE can be used immediately or aliquoted. Aliquots should be flash frozen in liquid nitrogen and stored at -80 °C. The NE can be stored and undergo at least two freeze-thaw cycles without losing activity.

8. Representative Results

Recently, efficient in vitro systems for coupling RNAP II transcription to splicing were developed^2-5. These systems employed HeLa cells grown in bulk and thus are not amenable to rapidly testing the effects of specific cellular treatments on multiple samples. Based on this need and the general utility of a small-scale extract protocol (see Discussion), we established a robust small-scale nuclear extract method. Representative data comparing the RNAP II transcription/splicing reaction in the small-scale nuclear extract with the bulk nuclear extract is shown in Figure 2. A CMV-DNA construct, which contains the CMV promoter and encodes a standard splicing substrate (Ftz³, Figure 2A) was used for the analysis. When this construct was incubated in the bulk (lanes 1-3) or small-scale (lanes 4-6) extract, similar levels of the nascent pre-mRNA were synthesized by the 5 min time point (Figure 2, lanes 1 and 4). Following addition of α-amanitin to block further transcription, the splicing intermediates and spliced products accumulated over time with similar kinetics in both types of extracts (Figure 2, lanes 2, 3, 5, 6). These representative results show that the efficiency of the coupled RNAP II transcription/splicing system is 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, E7107, or the negative control compound pladienolide F^6,7 and then the coupled RNAP II transcription/splicing assay was carried out. As shown in Figure 3, transcription by RNAP II occurred efficiently in extracts prepared from both the Pladienolide F and E7107 treated cells (10 min time points). In contrast, splicing occurred normally in the extract prepared from the Pladienolide F-treated cells but was abolished in the E7107-treated cells (Figure 3, 20-60 min time points). These data provide proof of concept for using the small-scale nuclear extracts for special treatment of cells in small scale.

Figure 1. Schematic of Small-scale Extract Protocol. Step P1. Cells are grown as monolayers, harvested from plates using a cell lifter and swollen by the addition of hypotonic buffer. Step P2. Cells are lysed using a Dounce homogenizer and centrifuged to pellet the nuclei. Step P3. Nuclei are separated from the cytoplasm and undergo salt extraction. Step P4. The nuclear extract is concentrated and dialyzed. Step P5. Results are obtained that show that nuclear extracts are functional (see Figure 2 for greater detail).

Figure 2. Small-scale nuclear extracts are robust in a coupled RNAP II transcription/splicing assay. A. Schematic of the CMV-Ftz DNA template used for coupled RNAP II transcription/splicing. The CMV promoter and the sizes of the exons and intron are indicated. B. Comparison of coupled RNAP II transcription/splicing assay using either bulk nuclear extract or small-scale nuclear extract. α-amanitin was added after 5 min of transcription and splicing was allowed to occur for 30 and 60 min. RNA was extracted and fractionated on a 5% denaturing polyacrylamide gel and detected by phosphoimager. The splicing intermediates and products are indicated. The endogenous U6 snRNA and tRNA present in the extract and that are ³²P-labeled during incubation are indicated. See³ for a detailed protocol on the coupled RNAP II transcription/splicing system.

Figure 3. Small-scale nuclear extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107 are defective in splicing. Cells were treated with 3 μM Pladienolide F or E7107 as described⁶ and then used to prepare small-scale nuclear extracts. A time course was carried out using the same transcription/splicing assay as in Figure 2. The splicing intermediates and products, and U6 snRNA and tRNA are indicated.