Name: small-scale nuclear extracts for functional assays of gene-expression machineries
Uploaded: 2012-06-27T13:00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries at JoVE.com

We are grateful to M. Winkelbauer-Hurt, E. Ibrahim, P. Valencia, K. Dufu, H. Cheng for useful discussions. HeLa cells were obtained from the National Cell Culture Center (Minneapolis, MN). We also thank Eisai Co., Ltd for providing E7107 and the Nikon Imaging Center at Harvard Medical School for help with light microscopy. This work was supported by an NIH grant GM043375 to RR and EGF and NRSA fellowship to JLH.

1. Growing HeLa Cells for Nuclear Extraction
<ol>
 <li>Plate HeLa cells on three 150 mm plates with DMEM media supplemented with 10% FBS and 1% Penicillin/Streptomycin. Grow in a 37 &deg;C incubator with 5% CO2 until cells are 90% confluent.</li>
</ol>
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
<ol>
 <li>Prepare fresh solutions as outlined in Table 2.</li>
 <li>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).</li>
</ol>
3. Harvesting HeLa Cells for Nuclear Extraction
<ol>
 <li>Aspirate media from the cells and wash the cells once with 13 ml of room temperature 1X PBS per plate.</li>
 <li>Aspirate the PBS wash.</li>
</ol>
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.
<ol start="3">
 <li>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.</li>
</ol>
Tip- It is easier to estimate the volume of cells if an Eppendorf tube is used. For scale up, use Falcon tubes.
<ol start="4">
 <li>Centrifuge at 4 &deg;C in a microfuge for 5 min at 100 x g to pellet the cells.</li>
</ol>
4. Swell HeLa Cells in Hypotonic Buffer
<ol>
 <li>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 &mu;l of PCV.</li>
 <li>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.</li>
 <li>Centrifuge at 4 &deg;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 &mu;l-1000 &mu;l.</li>
 <li>Carefully aspirate the Hypotonic Buffer and add new Hypotonic Buffer to a final volume of 1.5 ml. Resuspend the cells.</li>
 <li>Incubate the resuspended swollen cells on ice for 10 min.</li>
</ol>
5. Lyse Cells Using a Dounce Homogenizer
<ol>
 <li>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 &mu;l extended micropipette tip.</li>
 <li>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.</li>
 <li>Assay for cell lysis using a 5 &mu;l 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.</li>
</ol>
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.
<ol start="4">
 <li>Transfer the lysed cells to a pre-chilled Eppendorf tube and spin in a 4 &deg;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.</li>
</ol>
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
<ol>
 <li>Estimate the Packed Nuclear Volume (PNV) by looking at the gradation on the tube. A PCV of 500 &mu;l typically yields a PNV of 400 &mu;l.</li>
 <li>Add &frac12; 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.</li>
 <li>Add &frac12; X PNV of High Salt Buffer and quickly mix 1 time by inverting the tube. Do not shake. Rotate at 4 &deg;C for 30 min.</li>
</ol>
Tip- Be gentle with the nuclei when they are in High Salt Buffer to avoid lysing them.
<ol start="4">
 <li>Spin at 4 &deg;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 &mu;l of HS-NE.</li>
</ol>
7. Concentrate and Dialyze the Nuclear Extract
<ol>
 <li>Transfer 500 &mu;l of the HS-NE into chilled mini-centricons (Amicon) and spin for 50 min at 4 &deg;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 &deg;C 1000 x g to recover the HS-NE. The HS-NE will be concentrated to approximately 115 &mu;l.</li>
 <li>Using a P200 Pipetman, transfer 45 &mu;l 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 &deg;C. The NE will appear cloudy after dialysis.</li>
</ol>
Tip- Do not centrifuge to remove the cloudy precipitate that is observed after dialysis, as this centrifugation reduces the activity of the extract.
<ol start="3">
 <li>The NE can be used immediately or aliquoted. Aliquots should be flash frozen in liquid nitrogen and stored at -80 &deg;C. The NE can be stored and undergo at least two freeze-thaw cycles without losing activity.</li>
</ol>
8. Representative Results
Recently, efficient in vitro systems for coupling RNAP II transcription to splicing were developed2-5. 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 &alpha;-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 F6,7 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.
<img alt="Figure 1" src="/files/ftp_upload/4140/4140fig1.jpg" /> 
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).
<img alt="Figure 2" src="/files/ftp_upload/4140/4140fig2.jpg" />
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. &alpha;-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.
<img alt="Figure 3" src="/files/ftp_upload/4140/4140fig3.jpg" /> 
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 &mu;M 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.

<ol>
	<li>Krainer, A. R., Maniatis, T., Ruskin, B., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+and+mutant+human+beta-globin+pre-mRNAs+are+faithfully+and+efficiently+spliced+in+vitro.">Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.</a> Cell. 36, 993-1005 (1984).</li><li>Ghosh, S., Garcia-Blanco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupled+in+vitro+synthesis+and+splicing+of+RNA+polymerase+II+transcripts.">Coupled in vitro synthesis and splicing of RNA polymerase II transcripts.</a> Rna. 6, 1325-1334 (2000).</li><li>Das, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coupling+of+RNAP+II+transcription+to+spliceosome+assembly.">Functional coupling of RNAP II transcription to spliceosome assembly.</a> Genes Dev. 20, 1100-1109 (2006).</li><li>Hicks, M. J., Yang, C. R., Kotlajich, M. V., Hertel, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+splicing+to+Pol+II+transcription+stabilizes+pre-mRNAs+and+influences+splicing+patterns.">Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns.</a> PLoS Biol. 4, e147 (2006).</li><li>Yu, Y., Das, R., Folco, E. G., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+in+vitro+system+for+co-transcriptional+splicing.">A model in vitro system for co-transcriptional splicing.</a> Nucleic Acids Res. 38, 7570-7578 (2010).</li><li>Kotake, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+factor+SF3b+as+a+target+of+the+antitumor+natural+product+pladienolide.">Splicing factor SF3b as a target of the antitumor natural product pladienolide.</a> Nat. Chem Biol. 3, 570-575 (2007).</li><li>Folco, E. G., Coil, K. E., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-tumor+drug+E7107+reveals+an+essential+role+for+SF3b+in+remodeling+U2+snRNP+to+expose+the+branch+point-binding+region.">The anti-tumor drug E7107 reveals an essential role for SF3b in remodeling U2 snRNP to expose the branch point-binding region.</a> Genes. 25, 440-444 (2011).</li><li>Dignam, J. D., Lebovitz, R. M., Roeder, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+transcription+initiation+by+RNA+polymerase+II+in+a+soluble+extract+from+isolated+mammalian+nuclei.">Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.</a> Nucleic Acids Res. 11, 1475-1489 (1983).</li><li>Lee, K. A., Bindereif, A., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-scale+procedure+for+preparation+of+nuclear+extracts+that+support+efficient+transcription+and+pre-mRNA+splicing.">A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing.</a> Gene Anal. Tech. 5, 22-31 (1988).</li></ol>

Production and Free Access to this article is sponsored by Abcam, Plc.

We have established a rapid and reproducible method for preparation of nuclear extracts from small quantities of HeLa cells grown as monolayers. We demonstrated that these extracts are robust by showing that the kinetics and efficiency of RNAP II transcription/splicing assays are similar in the small-scale and bulk nuclear extracts. We showed the utility of the extracts by demonstrating that extracts prepared from cells treated with a splicing inhibitor drug are active for transcription but defective in splicing.
Our method for preparing small-scale nuclear extracts was established by combining and optimizing methods previously established for making extracts from HeLa cells grown in suspension in large-scale1,8 and a method for small-scale preparation of extracts9. We established our protocol because the previous small-scale extracts were not functional for some assays, such as the coupled transcription/splicing assay. An important difference between the previous small-scale protocol9 and ours is that we optimized conditions for lysing cells using a mini-dounce whereas the previous protocol lysed cells by pushing them through a small-gauge needle9. The needle-lysis results in bubbles, which may explain why the extracts were inactive and/or difficult to reproduce for some assays. We also added a concentration step to our protocol. This step increases the activity of the extracts, which are extremely sensitive to concentration, and also limits the variability between extracts that is inherent to small-scale preparations. Finally, our preparation is routinely carried out with only three 150 mm plates of monolayer cells, without any significant effect on activity compared to bulk extracts. Thus, the procedure is readily amenable for small-scale preparations that require costly reagents or limited cell availability. For example, we have prepared small-scale extracts from RNAi knockdown cells and from Chronic Lymphocytic Leukemia (CLL) patient cells, and used these extracts for functional and/or biochemical assays (EGF, JLH, TY and RR, unpublished). We have found that the extracts are valuable for RNAi followed by specific biochemical assays, such as immunopreciptations, Westerns, and silver staining. After establishing the efficacy of knocking down a particular protein, a more detailed study can be carried out by making a stable knockdown cell-line, which can be used to prepare additional extracts at lower cost. Mass spectrometry of proteins present in immunoprecipitates obtained from these knockdown cell lines will also be a useful application of the method. In addition to the coupled transcription/splicing assay that we used as an example for our protocol description here, the small-scale extracts should be generally applicable to numerous functional and biochemical assays, such as those used for the different steps in gene expression (e.g. capping, splicing, transcription, polyadenylation, microRNA processing). The protocol can also be adapted for patient cell types that can be either obtained in suspension (such as the CLL cells) or grown in culture (such as patient fibroblasts). Finally, the cytoplasmic fraction obtained during the procedure should prove useful for both functional and biochemical assays that require the cytoplasm.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Full name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td>
 <td>Sigma</td>
 <td>H3375-500G</td>
 </tr>
 <tr>
 <td>MgCl2.6H2O</td>
 <td>Magnesium Chloride Hexahydrate </td>
 <td>Sigma</td>
 <td>M2670-500G</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Potassium Chloride </td>
 <td>Fisher</td>
 <td>P335-212</td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>Phenylmethanesulfonyl fluoride</td>
 <td>Sigma</td>
 <td>P7626-100G</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Dithiothreitol</td>
 <td>American Bioanalytical</td>
 <td>AB00490-5G</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Ethylenediaminetetraacetate, disodium, dihydrate </td>
 <td>American Bioanalytical</td>
 <td>AB00500-01000</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>&nbsp;</td>
 <td>MP Biomedicals</td>
 <td>800689</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Dulbecco's Minimal Essential Medium</td>
 <td>Invitrogen</td>
 <td>11995-073</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Fetal Bovine Serum</td>
 <td>Gibco</td>
 <td>16140</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>&nbsp;</td>
 <td>Gibco</td>
 <td>15070</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Phosphate Buffered Saline</td>
 <td>Cellgro</td>
 <td>21-040-CV</td>
 </tr>
 <tr>
 <td>Trypan Blue Stain 0.4%</td>
 <td>&nbsp;</td>
 <td>Gibco</td>
 <td>15250</td>
 </tr>
 <tr>
 <td>Cell Lifters</td>
 <td>&nbsp;</td>
 <td>Corning</td>
 <td>29442-200</td>
 </tr>
 <tr>
 <td>150mm Plates</td>
 <td>&nbsp;</td>
 <td>VWR</td>
 <td>353025</td>
 </tr>
 <tr>
 <td>Extended Micropipette Tip</td>
 <td>&nbsp;</td>
 <td>Denville</td>
 <td>P1126</td>
 </tr>
 <tr>
 <td>Dounce homogenizer, 1ml, 
 with tight pestle</td>
 <td>&nbsp;</td>
 <td>Wheaton</td>
 <td>357538 </td>
 </tr>
 <tr>
 <td>Slidealyzers, MWCO 10kDa</td>
 <td>&nbsp;</td>
 <td>ThermoScientific</td>
 <td>69572</td>
 </tr>
 <tr>
 <td>Mini-Centricons, MWCO 3kDa</td>
 <td>&nbsp;</td>
 <td>Millipore (Amicon)</td>
 <td>UFC500396</td>
 </tr>
 </tbody>
</table>
Table 1. Specific Reagents and Equipment.
<table border="1">
 <tbody>
 <tr>
 <td>Solution</td>
 <td>Final</td>
 <td>Stock</td>
 <td>Dispense</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hypotonic</td>
 <td>10 mM HEPES, pH 7.9</td>
 <td>1M HEPES, pH 7.9</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>1.5 mM MgCl2</td>
 <td>1M MgCl2</td>
 <td>750 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>10 mM KCl</td>
 <td>3M KCl</td>
 <td>1.67 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.2 mM PMSF</td>
 <td>200 mM PMSF</td>
 <td>500 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.5 mM DTT</td>
 <td>2M DTT</td>
 <td>125 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bring up to 500 ml with water</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low Salt</td>
 <td>20 mM HEPES, pH 7.9</td>
 <td>1M HEPES, pH 7.9</td>
 <td>2 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>1.5 mM MgCl2</td>
 <td>1M MgCl2</td>
 <td>150 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>20 mM KCl</td>
 <td>3M KCl</td>
 <td>667 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.2 mM EDTA</td>
 <td>0.5M EDTA</td>
 <td>40 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>25% Glycerol</td>
 <td>Glycerol</td>
 <td>25 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.2 mM PMSF</td>
 <td>200 mM PMSF</td>
 <td>100 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.5 mM DTT</td>
 <td>2M DTT</td>
 <td>25 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bring up to 100 ml with water</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>High Salt</td>
 <td>20 mM HEPES, pH 7.9</td>
 <td>1M HEPES, pH 7.9</td>
 <td>2 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>1.5 mM MgCl2</td>
 <td>1M MgCl2</td>
 <td>150 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>1.4M KCl</td>
 <td>3M KCl</td>
 <td>48 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.2 mM EDTA</td>
 <td>0.5M EDTA</td>
 <td>40 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>25% Glycerol</td>
 <td>Glycerol</td>
 <td>25 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.2 mM PMSF</td>
 <td>200 mM PMSF</td>
 <td>100 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.5 mM DTT</td>
 <td>2M DTT</td>
 <td>25 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bring up to 100 ml with water</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dialysis</td>
 <td>20 mM HEPES, pH 7.9</td>
 <td>1M HEPES, pH 7.9</td>
 <td>10 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>100 mM KCl</td>
 <td>3M KCl</td>
 <td>16.625 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.2 mM EDTA</td>
 <td>0.5M EDTA</td>
 <td>200 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>20% Glycerol</td>
 <td>Glycerol</td>
 <td>100 ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.2 mM PMSF</td>
 <td>200 mM PMSF</td>
 <td>500 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.5 mM DTT</td>
 <td>2M DTT</td>
 <td>125 &mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bring up to 500 ml with water</td>
 </tr>
 </tbody>
</table>
Table 2. Solutions for Nuclear Extract Preparation.

small-scale nuclear extracts for functional assays of gene-expression machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

Watch this Scientific Journal Video about Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries at JoVE.com

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

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

small-scale-nuclear-extracts-for-functional-assays-gene-expression

Research

JoVE Journal

Biology

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...