This work was in part supported by NCI (CA193549) and CCHMC Research Innovation Pilot awards to Kakajan Komurov, and Department of Defense (BC150484) award to Navneet Singh. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the Department of Defense. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The Institutional Animal Care and Use Committee and Institutional Biosafety Committee of the Cincinnati Children&#8217;s Research Foundation approved all animal experimental procedures (IACUC protocol #2017-0061 and IBC protocol #IBC2016-0016), and these experiments were carried out in accordance with standards as described in the NIH Guide to the Care and Use of Laboratory Animals.
1. Chronic inhibition of RNA Pol II by flavopiridol treatment&#8212;basic strategy
<ol>
	<li>Seed B16/F10 mouse melanoma cells in low density (0.2 x 106) in a 10 cm culture plate in their corresponding medium (Dulbecco&#8217;s Modified Eagle Medium [DMEM], 10% fetal bovine serum [FBS], 1% penicillin and streptomycin [Pen/Strep]) and incubate overnight in a 37 &#176;C, 5% CO2 humidified incubator.</li>
	<li>Following day, wash the cells with 1x phosphate-buffered saline (PBS) and add a new batch of culture media with a sublethal dose (estimated as 25&#8201;nM) of flavopiridol&#8212;an inhibitor of the RNA Pol II elongation factor p-TEFb (cyclin T/CDK9)&#8212;for one week without further sub-culturing.</li>
	<li>Following a week of flavopiridol treatment, perform confirmatory assays to evaluate the model&#8217;s ability to recapitulate various attributes of transcriptional elongation defects seen in TEdeff cancers.</li>
</ol>
2. Confirmatory immunoblot assay to assess defective RNA Pol II function and impairment of interferon (IFN) pathway and tumor necrosis factor (TNF) pathway signaling of in the generated mouse TEdeff model
<ol>
	<li>Culture equal number (105) of flavopiridol treated B16/F10 mouse melanoma cells and parental B16/F10 mouse melanoma cells in two different sets of 12 well plates (one set for RNA Pol II functional characterization and the other set for cytokine stimulation) at 37 &#176;C in a 5% CO2 humidified incubator overnight.</li>
	<li>Next day, treat the cells in the cytokine stimulation set with mouse IFN-&#947;, IFN-&#945; (5&#8201;ng/mL), or TNF-&#945; (5&#8201;ng/mL) for 45&#8201;min at 37 &#176;C.</li>
	<li>Now, extract protein from cells in both cytokine and RNA Pol II functional characterization sets using a radioimmunoprecipitation assay (RIPA) lysis buffer in the following manner:
	<ol>
		<li>Wash cells with 1x PBS and lyse it with 50 &#181;L of the lysis buffer per well. Scrape, and then pellet the lysed cells at 4 &#176;C, 21,130 x g.</li>
		<li>Measure the protein in cell lysate supernatants using a standard colorimetric assay for protein concentration following detergent solubilization (Bradford or a similar assay).</li>
	</ol>
	</li>
	<li>Load equal amount of measured protein (15&#8201;&#181;g) from each sample to run in a 4%&#8722;18% sodium dodecyl sulfate (SDS) polyacrylamide gel, and transfer them onto polyvinylidene difluoride (PVDF) membranes.</li>
	<li>Block the PVDF membranes in 5% dry milk in tris-buffered saline&#8722;polysorbate 20 (TBST) for 1&#8201;h followed by an overnight incubation at 4 &#176;C with primary antibodies (RNA Pol II 1:1000; p-SER2 RNA Pol II 1:1000; p-SER5 RNA Pol II 1:1000; H3K36me3 1:2000; total H3 1:2000; STAT1 1:1000; p-STAT1 1:1000; NF&#954;B 1:1000; p-NF&#954;B 1:1000; &#946;-Actin 1:5000) in 5% bovine serum albumin.</li>
	<li>Following day, wash the PVDF membranes with 1x TBST for 15 min at room temperature (RT), and incubate them with appropriate secondary antibodies (anti-rat [1:5000] for RNA Pol II, p-SER2 RNA Pol II, and p-SER5 RNA Pol II; anti-rabbit (1:5000) for H3K36me3, total H3, STAT1, p-STAT1, NF&#954;B, and p-NF&#954;B) for 50 min at RT. Detect the protein signals using commercially available horseradish peroxidase (HRP) substrate with enhanced chemiluminescence. 
	NOTE: &#946;-Actin primary used is HRP-conjugated, therefore it can be developed without a secondary.</li>
</ol>
3. Confirmatory assay to assess mRNA processing defects in the generated mouse TEdeff model
<ol>
	<li>Seed equal number (0.2 x 106) of flavopiridol treated B16/F10 mouse melanoma cells and parental B16/F10 mouse melanoma cells in 6-well plates at 37 &#176;C in a 5% CO2 humidified incubator overnight.</li>
	<li>Extract total RNA from the cultured cells at 60% confluency using an RNA extraction reagent or kit (Table of Materials).</li>
	<li>Deplete rRNA from the total extracted RNA in the following manner: 
	NOTE: A low-input protocol has been co-opted from a commercially available kit to deplete rRNA
	<ol>
		<li>Set one water bath or heat block to 70&#8722;75 &#176;C, and another water bath or heat block at 37 &#176;C.</li>
		<li>Add the total RNA (100&#8722;500 ng in 2 &#181;L of nuclease-free water) with 1 &#181;L of selective rRNA depletion probe and 30 &#181;L of hybridization buffer in a microcentrifuge tube, mix gently by vortexing and incubate them at 70&#8722;75 &#176;C for 5 min.</li>
		<li>Now, transfer the tubes to a 37 &#176;C water bath/heat block, and allow the sample to cool to 37 &#176;C over a period of 30 min.</li>
		<li>Resuspend the selective rRNA depletion probe magnetic beads by vortexing, and aliquot 75 &#181;L of beads in a 1.5 mL RNase-free microcentrifuge tube.</li>
		<li>Place the bead suspension on a magnetic separator for 1 min. Allow the beads to settle. Gently aspirate and discard the supernatant. Repeat washing the beads once again by adding 75 &#181;L of nuclease-free water and discarding the supernatant following magnetic separation.</li>
		<li>Resuspend the washed beads in 75 &#181;L of hybridization buffer, and aliquot 25 &#181;L of it to another tube and maintain it at 37 &#176;C for later use.</li>
		<li>Place the remaining 50 &#181;L beads on a magnetic separator for 1 min and discard the supernatant. Resuspend the beads in 20 &#181;L of hybridization buffer and maintain it at 37 &#176;C for later use.</li>
		<li>After the cooling of the RNA/selective rRNA depletion probe mixture to 37 &#176;C for 30 min, briefly centrifuge the tube to collect the sample to the bottom of the tube.</li>
		<li>Transfer 33 &#181;L of the RNA/selective rRNA depletion probe mixture to the prepared magnetic beads from step 3.3.7. Mix by low speed vortexing.</li>
		<li>Incubate the tube at 37 &#176;C for 15 min. During incubation, gently mix the contents occasionally. Followed by brief centrifugation to collect the sample to the bottom of the tube.</li>
		<li>Place the tube on a magnetic separator for 1 min to pellet the rRNA-probe complex. This time do not discard the supernatant. The supernatant contains rRNA-depleted RNA.</li>
		<li>Place the tube of 25 &#956;L of beads from step 3.3.6 on a magnetic separator for 1 min. Aspirate and discard the supernatant. Add the supernatant from step 3.3.11 to the new tube of beads. Mix by low speed vortexing.</li>
		<li>Incubate the tube at 37 &#176;C for 15 min. During incubation, gently mix the contents occasionally. Briefly centrifuge the tube to collect the sample to the bottom of the tube.</li>
		<li>Place the tube on a magnetic separator for 1 min to pellet the rRNA-probe complex. Do not discard the supernatant. Transfer the supernatant (about 53 &#956;L) containing rRNA-depleted RNA to a new tube.</li>
		<li>Measure the concentration of the RNA yield by a spectrophotometer.</li>
	</ol>
	</li>
	<li>Use one half of the rRNA-depleted samples as input to magnetic beads containing oligo (dT)25 to extract polyA+&#8201;RNA in the following manner: 
	NOTE: This protocol of isolating polyA tail messenger RNA using oligo dT sequences bound to the surface of magnetic beads has been co-opted from a commercially available kit (Table of Materials).
	<ol>
		<li>Resuspend the oligo dT beads in the vial by briefly vortexing for &#62;30 s and transfer 200 &#181;L of oligo dT beads to a tube. Add the same volume (200 &#181;L) of binding buffer, and resuspend.</li>
		<li>Place the tube in a magnet for 1 min and discard the supernatant. Now, remove the tube from the magnet and resuspend the washed oligo dT beads in 100 &#181;L of binding buffer.</li>
		<li>Adjust the volume of the input rRNA-depleted total RNA sample to 100 &#956;L with 10 mM Tris-HCl pH 7.5. Now, add 100 &#956;L of binding buffer.</li>
		<li>Heat to 65 &#176;C for 2 min to disrupt secondary RNA structures. Now, immediately place on ice.</li>
		<li>Add the 200 &#956;L of total RNA to the 100 &#956;L washed beads. Mix thoroughly and allow binding by rotating continuously on a rotor for 5 min at RT.</li>
		<li>Place the tube on the magnet for 1-2 min and carefully remove all the supernatant and carefully remove all supernatant.</li>
		<li>Remove the tube from the magnet and add 200 &#956;L of Washing Buffer.</li>
	</ol>
	</li>
	<li>Measure the purity and concentration of the extracted polyA+ RNA by a spectrophotometer. 
	NOTE: A 260/280 ratio of 1.90&#8722;2.00, and a 260/230 ratio of 2.00&#8722;2.20 for all RNA samples are considered acceptable.</li>
	<li>Use the remaining half of the rRNA-depleted samples from section 3.3 as input to protein A columns (provided in the RNA immunoprecipitation [RIP] kit, Table of Materials) to immunoprecipitate five-prime capped RNAs using monoclonal 7-methylguanosine antibody in the following manner: 
	NOTE: This protocol of isolating m7G capped messenger RNA using a commercially available RNA immunoprecipitation kit has been co-opted and further modified.
	<ol>
		<li>Wash the protein A magnetic beads obtained from the RIP kit according to manufacturer&#8217;s protocol to pre-bind the antibody to the beads.</li>
		<li>Transfer 3 &#181;g of 7-methylguanosine antibody (rabbit IgG provided in the kit can be used the negative control) to the beads in a microcentrifuge tube suspended in 100 &#181;L wash buffer from the kit.</li>
		<li>Incubate with low speed rotation for 30 min at RT. Centrifuge tubes briefly and then place the tubes on a magnetic separator, remove and discard the supernatant.</li>
		<li>Remove tubes and add 500 &#181;L of wash buffer from the kit and vortex briefly. Centrifuge the tubes briefly followed by magnetic separation once again, remove and discard the supernatant.</li>
		<li>Repeat step 3.6.4 once again.</li>
		<li>Add around 120 ng of rRNA depleted (from section 3.3) to the prewashed 7-methylguanosine antibody bound beads. Add 1 &#181;L of RNase inhibitor. Incubate at RT for 1&#8722;1.5 h with mild agitation.</li>
		<li>Spin down the beads at 300 x g for 10 s and remove the supernatant containing uncapped (non-7-methylguanosine) mRNA to a new microcentrifuge tube.</li>
		<li>Add 100 &#181;L of wash buffer and wash it two more times similarly. Pool the collected supernatant in the same microcentrifuge tube labelled uncapped (non-7-methylguanosine) mRNA. Store on ice.</li>
		<li>Elute the capped (7-methylguanosine) mRNA from the beads with 300 &#181;L of urea lysis buffer (ULB) containing 7 M urea, 2% SDS, 0.35 M NaCl, 10 mM EDTA and 10 mM Tris, pH 7.5 by heating the beads at 65 &#176;C for 2&#8722;3 min.</li>
		<li>Mix 300 &#181;L of the eluted samples (capped and uncapped mRNA) with 300 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1; commercially available) (stored at 4 &#176;C). Mix well by inverting and leave for about 10 min then mix again gently.</li>
		<li>Centrifuge at 18,928 x g for 2 min and carefully pipette the top layer to fresh tube and discard bottom layer.</li>
		<li>Add 300 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1; stored at 4 &#176;C) to the samples. Mix well by inverting and then centrifuge at 18,928 x g for 1 min. Carefully, pipette the top layer to a fresh tube and discard bottom layer.</li>
		<li>Add 300 &#181;L of 2-porpanol and 30 &#181;L of 3 M sodium acetate (pH 5.2) to the capped and uncapped RNA. Invert the sample a few times and put it at -20 &#176;C for 20 h.</li>
		<li>Now, centrifuge the samples at 18,928 x g for 10 min at 4 &#176;C. Carefully remove the supernatant and add 500 &#181;L of 70% ethanol.</li>
		<li>Centrifuge again at 18,928 x g for 10 min at 4 &#176;C. Carefully discard the supernatant and dry the pellet at RT for less than 5 min. Resuspend the pellet in nuclease-free water.</li>
	</ol>
	</li>
	<li>Measure the purity and concentration of RNA yield by a spectrophotometer. The 260/280 ratio should be in the range of 1.90&#8722;2.00, and the 260/230 ratio in the range of 2.00&#8722;2.20 for all RNA samples.</li>
</ol>
4. Confirmatory assay to assess the response of mouse TEdeff model to FasL mediated cell death
<ol>
	<li>Seed equal number (30,000 cells) of flavopiridol treated B16/F10 mouse melanoma cells and parental B16/F10 mouse melanoma cells in a 96-well culture plate in their corresponding medium (DMEM), and incubate overnight in a 37 &#176;C, 5% CO2 humidified incubator.</li>
	<li>Treat the cells in a culture hood with different concentrations of hhis6FasL (0.1&#8722;1000&#8201;ng/mL) in the presence of 10&#8201;&#956;g/mL anti-His antibody and incubate for 24&#8201;h at 37 &#176;C, 5% CO2 humidified incubator.</li>
	<li>Remove dead cells by washing with 1x PBS buffer. Fix the attached cells in 4% paraformaldehyde for 20 min at RT. Discard the 4% paraformaldehyde (no need to wash), and stain with crystal violet solution (20% methanol, 0.5% crystal violet in 1x PBS) for 30&#8201;min.</li>
	<li>Remove excess stain by gently rinsing the plates in tap water. Keep the plates to dry at RT.</li>
	<li>Re-dissolve the crystal violet in 100 &#181;L of 1x nonionic surfactant dissolved in 1x PBS, and measure cell density by measuring the absorbance at 570&#8201;nm in a microplate reader.</li>
</ol>
5. Exploratory assay to assess the response of mouse TEdeff model to antigen specific cytotoxic T-cell attack
<ol>
	<li>Isolation and activation of OT-I CD8+ cytotoxic T-cell attack (CTL)
	<ol>
		<li>Purify CD8+ cells from spleens of OT-I TCR Tg RAG-1&#8722;/&#8722; mice by magnetic cell separation using a mouse CD8 T cell isolation kit as follows:
		<ol>
			<li>Harvest two spleens from two OT-I TCR Tg RAG-1&#8722;/&#8722; mice in complete RPMI media.</li>
			<li>Mash the spleens in a 70 &#181;m filter kept on a 50 mL tube filled with 20 mL of RPMI, using the back of a syringe till only fat is left behind in the filter.</li>
			<li>Centrifuge the flow through at 220 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
			<li>Add 1 mL of red blood cell (RBC) lysis buffer to the spleen pellet from the previous centrifugation step and pipette the mixture for 1 min.</li>
			<li>Neutralize the solution by adding up to 10 mL of RPMI.</li>
			<li>Centrifuge at 220 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend in 10 mL of RPMI.</li>
			<li>Take a small aliquot for counting. Centrifuge the remaining at 220 x g for 5 min at 4 &#176;C.</li>
			<li>For every million cells counted, resuspend the pellet in 1 mL of commercially available magnetic separation system buffer (Mojo buffer or a similar buffer).</li>
			<li>Prepare an antibody cocktail of volume 100 &#181;L for every 1 mL of pelleted cells in step 5.1.1.8. The antibody cocktail includes: biotin anti-CD4, CD105, CD45R/B220, CD11c, CD49b, TER-119, CD19, CD11b, TCR &#947;/&#948;, and CD44.</li>
			<li>Add this cocktail to the 1 mL pelleted cells and keep on ice for 15 min.</li>
			<li>Add 100 &#181;L of magnetic (streptavidin) beads to every 100 &#181;L of the antibody cocktail added to the 1 mL resuspended pelleted spleen cells. Keep on ice for 15 min.</li>
			<li>Add 7 mL of commercially available magnetic separation system buffer. Now, aliquot about 3&#8722;4 mL of the mixture to a fresh tube. Mix well and fix it to the magnet for 5 min.</li>
			<li>Decant the liquid (contains CD8+ cells) to a fresh tube on ice. Now, aliquot the remaining 3&#8722;4 mL of mixture from step 5.1.1.12 to the tube and fix it to the magnet for 5 min. Decant the liquid (second batch of CD8+ cells isolated) to the same tube containing the first batch of CD8+ cells kept on ice.</li>
		</ol>
		</li>
		<li>Seed engineered adherent fibroblast APC&#173;-(MEC.B7.SigOVA) line to express a specific ovalbumin (OVA)-derived, H-2Kb-restricted peptide epitope OVA257-264 (SIINFEKL), along with the co-stimulatory molecule B7.1, at 75,000 cells per well in 24-well plates, at 37 &#176;C, 5% CO2 humidified incubator. 
		NOTE: The adherent fibroblast used is a gift from Dr. Edith Janssen&#8217;s lab at CCHMC. The line was created originally in Dr. Stephen P. Schoenberger&#8217;s lab in La Jolla Institute for Allergy and Immunology6.</li>
		<li>After 24&#8201;h, wash the monolayer of APC once with Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM) commercially available with HEPES buffer, sodium pyruvate, L-glutamine and high glucose), and add 0.5&#8201;x&#8201;106 naive OT-I CD8+ cells (from step 5.1.1.13) in 2&#8201;mL of IMDM supplemented with 50&#8201;mM &#946;-ME, 2&#8201;mL EDTA, 4&#8201;mM L-glutamine and HEPES and 10% FBS.</li>
		<li>After 20&#8201;h, gently harvest the non-adherent OT-I cells (by collecting the media in the culture dish with floating OT-I cells and pelleting the cells at 191 x g for 2 min; count the viable OT-I cells) and transfer them for co-culture.</li>
	</ol>
	</li>
	<li>Co-culture of CD8+ cells with B16/F10-OVA cells
	<ol>
		<li>Seed OT-I-derived CD8+ cells at a ratio of 1:1 (300,000 cells each) in a co-culture with B16/F10 (lacking the antigen ovalbumin), untreated B16/F10-OVA, and B16/F10-OVA cells pre-treated with flavopiridol (25 nM) for 1 week, in 6-well dishes with complete DMEM media for 20&#8201;h at 37 &#176;C in a 5% CO2 humidified incubator.</li>
		<li>After 20 h, remove the OT-I-derived CD8+ cells (by collecting the media in the culture dish with floating OT-I derived CD8+ cells). Wash the adherent B16/F10-OVA cells in 1x PBS.</li>
		<li>Trypsinize the three groups of attached B16/F10-OVA cells in 0.05% EDTA containing trypsin for 5 min. Pellet the trypsinized cells at 191 x g for 5 min.</li>
		<li>Stain the harvested B16/F10-OVA cells by incubating them in cold PBS (containing 0.5% FBS and 0.05% sodium azide) with viability dye and relevant labeled antibodies (fixable viability staining dye e780, AF647-conjugated mouse CD8 and BV421-conjugated mouse CD45).</li>
		<li>Analyze the viability of the three groups of B16/F10-OVA cells by flow cytometry.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

RNA Pol II elongation control has emerged as a decisive lever for regulating stimulus-responsive gene expression to the benefit of malignant cells5,7,8. Overcoming promoter-proximal pausing to elongation and subsequent mRNA production requires the kinase activity of P-TEFb9,10,11. Our model utilizes flavopiridol (25 nM), an inhibitor of...

A key rate-limiting step in the expression of nearly all active genes is the transition of RNA polymerase II (RNA Pol II) from promoter-proximal pausing to productive elongation1,2. Given that epigenetic dysregulation of transcriptional elongation assists in the progression of multiple human malignancies defined as TEdeff, leading to suboptimal signaling in the pro-inflammatory response pathways amounting to a poor response and outcome to immunotherapy3, the overarching goal of this protocol is to establish a useful in vitro model to study these widespread non-genetic transcriptional abnormalities in cancers. In this light, the use of chronic pharmacological inhibition of CDK9 is an efficacious strategy for creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects. The rationale behind using chronic CDK9 inhibition is that it abrogates phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA Pol II, thus repressing the release of RNA Pol II into productive transcription elongation. Also, TEdeff cancers, described previously by our group3, are not classified under any specific somatic mutation. Therefore, a non-genetic (pharmacological) model is advantageous and best mimics the widespread transcriptional and epigenetic defects observed in them. The method herein details the generation and characterization of chronic flavopiridol treatment model of murine cancer cells. This method demonstrably disrupts transcription elongation along genes characterized by longer genomic lengths, with poised promoters and inducible expressions such as TNF/NF-&#954;B and interferon/STAT signaling, profoundly controlled at the level of transcription elongation3,4,5. Overall, this optimized murine cell line model of transcriptional elongation defects&#8212;the only model to our knowledge to study the newly described TEdeff tumors&#8212;drives resistance to anti-tumor immune attack, rendering a useful system to exploit and examine the vulnerabilities of non-genetic defects in core transcription machinery in cancers vis-&#224;-vis immune-mediated cell attack.

<table><tbody><tr><td>hhis6FasL</td><td>Cell Signaling</td><td>5452</td><td></td></tr><tr><td>10X TBS</td><td>Bio-Rad</td><td>170-6435</td><td></td></tr><tr><td>12 well plates</td><td>Falcon</td><td>353043</td><td></td></tr><tr><td>20% methanol</td><td>Fisher Chemical</td><td>A412-4</td><td></td></tr><tr><td>24-well plates</td><td>Falcon</td><td>351147</td><td></td></tr><tr><td>4&amp;ndash;18% SDS polyacrylamide gel</td><td>Bio-Rad</td><td>4561086</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Thermo Fisher Scientific</td><td>AAJ19943K2</td><td></td></tr><tr><td>5% dry milk</td><td>Bio-Rad</td><td>170-6404</td><td></td></tr><tr><td>7-Methylguanosine antibody</td><td>BioVision</td><td>6655-30T</td><td></td></tr><tr><td>96-well plates</td><td>Cellstar</td><td>655180</td><td></td></tr><tr><td>AF647-conjugated mouse CD8</td><td>Biolegend</td><td>100727</td><td></td></tr><tr><td>antibiotic and antimycotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>anti-His antibody</td><td>Cell Signaling</td><td>2366&amp;thinsp;P</td><td></td></tr><tr><td>Anti-Rabit</td><td>Cell Signaling</td><td>7074</td><td>Dilution 1:5000</td></tr><tr><td>Anti-Rat</td><td>Cell Signaling</td><td>7077S</td><td>Dilution 1:5000</td></tr><tr><td>Bradford assay Kit</td><td>Bio-Rad</td><td>5000121</td><td></td></tr><tr><td>BSA</td><td>ACROS Organics</td><td>24040-0100</td><td></td></tr><tr><td>BV421-conjugated mouse CD45</td><td>Biolegend</td><td>109831</td><td></td></tr><tr><td>crystal violet</td><td>Sigma</td><td>C3886-100G</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>Dynabeads Oligo (dT)25</td><td>Ambion</td><td>61002</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>45015</td><td></td></tr><tr><td>Fixable Live/Dead staining dye e780</td><td>eBioscience</td><td>65-0865-14</td><td></td></tr><tr><td>Flavopiridol</td><td>Selleckchem</td><td>S1230</td><td></td></tr><tr><td>H3k36me3</td><td>Abcam</td><td>ab9050</td><td>Dilution 1:2000</td></tr><tr><td>IFN-&amp;alpha;</td><td>R&amp;amp;D systems</td><td>12100-1</td><td></td></tr><tr><td>IFN-&amp;gamma;</td><td>R&amp;amp;D systems</td><td>485-MI-100</td><td></td></tr><tr><td>IMDM</td><td>Gibco</td><td>12440053</td><td></td></tr><tr><td>Immobilon Western Chemiluminescent HRP Substrate</td><td>Millipore</td><td>WBKLS0500</td><td></td></tr><tr><td>MojoSort Mouse CD8 T Cell Isolation Kit</td><td>Biolegend</td><td>480007</td><td></td></tr><tr><td>NF-&amp;kappa;B</td><td>Cell Signaling</td><td>8242s</td><td>Dilution 1:1000</td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>p-NF-&amp;kappa;B</td><td>Cell Signaling</td><td>3033s</td><td>Dilution 1:1000</td></tr><tr><td>p-Ser2-RNAPII</td><td>Active Motif</td><td>61083</td><td>Dilution 1:500</td></tr><tr><td>p-Ser5-RNAPII</td><td>Active Motif</td><td>61085</td><td>Dilution 1:1000</td></tr><tr><td>p-STAT1</td><td>Cell Signaling</td><td>7649s</td><td>Dilution 1:1000</td></tr><tr><td>RiboMinu Eukaryote Kit</td><td>Ambion</td><td>A10837-08</td><td></td></tr><tr><td>RIPA buffer</td><td>Santa Cruz Biotechnology</td><td>sc-24948</td><td></td></tr><tr><td>RNAPII</td><td>Active Motif</td><td>61667</td><td>Dilution 1:1000</td></tr><tr><td>STAT1</td><td>Cell Signaling</td><td>9175s</td><td>Dilution 1:1000</td></tr><tr><td>TNF-&amp;alpha;</td><td>R&amp;amp;D systems</td><td>410-MT-010</td><td></td></tr><tr><td>total H3</td><td>Cell Signaling</td><td>4499</td><td>Dilution 1:2000</td></tr><tr><td>Tri reagent</td><td>Sigma</td><td>T9424</td><td></td></tr><tr><td>Triton</td><td>Sigma</td><td>T8787-50ML</td><td></td></tr><tr><td>Tween 20</td><td>AA Hoefer</td><td>9005-64-5</td><td></td></tr><tr><td>&amp;beta;-Actin</td><td>Cell Signaling</td><td>12620S</td><td>Dilution 1:5000</td></tr><tr><td>&amp;beta;-ME</td><td>G Biosciences</td><td>BC98</td><td></td></tr></tbody></table>

a murine cell line based model of chronic cdk9 inhibition to study widespread non-genetic transcriptional elongation defects (tedeff) in cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

Here, we provide a detailed scheme (Figure 1) to establish a TEdeff cell model obtained by chronic sub-lethal (Figure 2) treatment with flavopiridol at 25 nM. In Figure 3, on 3 days of treatment with flavopiridol, B16 OVA cells show partial characteristics of TEdeff but after one week of treatment, B16/F10 OVA cells show a profound loss of phosphorylation at serine 2 position on...

Watch this Scientific Journal Video about A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers at JoVE.com

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

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Research

JoVE Journal

Genetics

5.5K Views.  Cincinnati Children's Hospital Medical Center (CCHMC). The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

Video: A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Cancer Research

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Medicine

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Biology

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE^deff) in Cancers

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