The authors thank Barbara Bleekmann for excellent technical assistance. These studies were supported by the IFORES-program of the University of Duisburg-Essen Medical School and the RIMUR-program of the University Alliance Ruhr and Mercator Research Center Ruhr (MERCUR). The authors thank the J&#252;rgen-Manchot-Stiftung for the doctoral fellowship of Helene Sertznig and constant support. The collection and use of patient material has been approved by the ethics committee of the medical faculty of the University Duisburg-Essen (14-6028-BO).

This protocol follows the guidelines of human research as approved by the ethic committee of the medical faculty of the University of Duisburg-Essen (14-6028-BO).
1. Collection of blood or urine samples and isolation of polyomavirus DNA
<ol>
	<li>&#160;Collect at least 3 mL of blood in EDTA tubes or urine in acquisition tubes.</li>
	<li>Centrifuge the sample at 2,500 x g for 15 min. If needed, pipette plasma into a new tube and store the plasma samples at 4 &#176;C for several days or freeze at -20 &#176;C for longer storage.</li>
	<li>Prepare 40 &#181;L of proteinase K into a 1.5 mL microcentrifuge tube and add 400 &#181;L of plasma by pipetting.</li>
	<li>Lyse the sample by adding 400 &#181;L of lysis buffer, vortex for 15 s, and incubate for 10 min at 56 &#176;C.</li>
	<li>Isolate the DNA using a DNA blood extraction kit as described in the manufacturer&#39;s instructions. Briefly, add alcohol and load the lysates onto a spin column containing a DNA binding silica-based membrane. Wash the columns several times to yield pure DNA.</li>
	<li>Elute the yielded DNA in 30-50 &#181;L of TE buffer.</li>
</ol>
2. Amplification of the non-coding control region (NCCR)
<ol>
	<li>Perform the following procedure in physically separated rooms. Use an isolated room to prepare the reagents and distribute the mix to the PCR tubes.</li>
	<li>Prepare the Master Mix for the pre-PCR using primer pair A (Table 1) in a total volume of 50 &#181;L and use the previously isolated DNA from step 1.6. The pipetting scheme for preparing the master mix for the pre and nested PCR is printed in Table 2.</li>
	<li>Distribute 45 &#181;L of the master mix into the PCR tubes.</li>
	<li>Add 5 &#181;L of the isolated DNA into the PCR tubes. 
	NOTE: Use another room than the one for the master mix preparation to avoid contamination.</li>
	<li>Run the PCR with the reaction conditions as illustrated in Table 3. Repeat denaturation, annealing, and extension in 35 cycles.</li>
	<li>For the nested amplification use primer pair B harboring the restriction sites for AgeI and SpeI (Table 1).</li>
	<li>Run the nested PCR with the reaction conditions as described in steps 2.2 to 2.5 using 5 &#181;L of the pre-PCR.</li>
	<li>Mix 10 &#181;L of the PCR product with 2 &#181;L of 6x gel loading dye. Load 10 &#181;L of the mix on a 1.5% agarose gel, and run the gel for 30 min at 60 mA.</li>
	<li>Visualize the gel using an appropriate UV documentation system. 
	NOTE: The size of the amplicon is expected to be between 300-500 bp depending on whether rearrangements (deletions, insertions, or duplications) occurred (Figure 2C).</li>
	<li>Purify the PCR amplicons using a PCR purification kit according to the manufacturer&#39;s instructions. Elute the PCR amplicons in 30 &#181;L of elution buffer.</li>
	<li>Send the amplicons for Sanger sequencing using primer pair B.</li>
</ol>
3. Cloning of the NCCR into the dual fluorescence reporter
<ol>
	<li>Digest the purified amplicons (step 2.10) with AgeI and SpeI for 2 h at 37 &#176;C as described in Table 4.</li>
	<li>Repeat step 2.10 and purify the digested amplicons.</li>
	<li>In parallel, also digest the plasmid backbone (1.5 &#181;g) with AgeI and SpeI for 2 h at 37 &#176;C as described in Table 5. This step only needs to be performed once. For subsequent reactions, freeze the digestion at -20 &#176;C.</li>
	<li>Analyze the digested plasmid backbone on a 0.8% low melt agarose gel. 
	NOTE: Do not run the gel with a current higher than 40 mA. The expected insert of the spacer region originally derived from the plasmid pEX-K4 2-LTR CD313 is 128 bp (Figure 2C).</li>
	<li>Visualize DNA fragments using long-wave UV light (320 nm) and cut out the backbone band using a clean scalpel. Transfer the low melt gel fragment into a new 1.5 mL microcentrifuge tube. Avoid long UV exposure times to prevent DNA damage. 
	NOTE: Since low melt agarose is used, it is not necessary to purify the DNA before ligation.</li>
	<li>Heat the backbone containing the low melt agarose piece for 10 min at 65 &#176;C in order to melt the gel piece and mix every 2 min by gentle vortexing. The melted gel can be directly used for ligation.</li>
	<li>Ligate the backbone and the digested amplicon using T4 DNA ligase over night at 16 &#176;C using the scheme shown in Table 6.</li>
	<li>Perform transformation in E. coli using the heat shock method, plate bacteria on LB-amp plates, and culture at 37 &#176;C over night.</li>
	<li>On the next day, select three positive clones and prepare overnight E. coli cultures (5 mL of LB-Amp) and culture at 37 &#176;C.</li>
	<li>Isolate the plasmid-DNA using standard protocols as described elsewhere14, perform AgeI and SpeI digestion to check for positive clones and visualize cut out fragments on a 1% agarose gel. The spacer band (128 bp) will be replaced by the larger NCCR-sequence (300-500 bp, see above).</li>
	<li>Send the plasmid for Sanger sequencing using primer EGFP-N or primer pair B (Table 1).</li>
	<li>Since mutations might spontaneously occur, compare the sequencing results with the sequencing results obtained with the amplicon. Only use the clones containing identical sequences compared to the amplicon.</li>
	<li>Use a molecular workbench software (e.g., freeware GENtle 1.9.4 or other sequence editing programs) to align and edit the obtained sequences (Figure 3).</li>
	<li>Start GENtle 1.9.4 and import the DNA sequences by clicking on the Import button (green down arrow).</li>
	<li>Next click on Tool and select Alignment from the menu (Use Ctrl+G as a shortcut) and choose the DNA sequences for alignment.</li>
	<li>Add a sequence to the alignment by clicking on Add or remove a sequence by clicking on Remove. Include an archetypical NCCR consensus sequence like JN19243815, do not use the NCCR-sequence from the commonly used Dunlop-strain16, since it harbors rearrangements and duplications others than the archetypical BKPyV strains. 
	NOTE: The NCCR already contains the translational start codon (Figure 3).</li>
	<li>Choose a method for the alignment by clicking on Algorithm in the toolbar and choose clustal W. Set the alignment parameters to match 2; gap extension penalty -1; gap penalty -2 and click on OK to run the alignment.</li>
</ol>
4. Transient transfection of HEK293T cells with the reporter plasmid and treatment with potential antiviral agents
<ol>
	<li>To prepare sufficient amounts of plasmid DNA for subsequent transfection experiments prepare a 150 mL overnight culture. Isolate the plasmid DNA using a plasmid isolation kit. Alternatively, other plasmid purification kits may be used.</li>
	<li>Seed 1 x 105 HEK293T cells in DMEM containing 10% FCS, penicillin and streptomycin (1x) per well of a 12-well plate 24 h prior to transfection and incubate overnight at 37 &#176;C and 5% CO2 to maintain active proliferation during transfection. 
	NOTE: Cells should be approximately 80% confluent at transfection.</li>
	<li>Place 250 &#181;L of reduced serum media in a sterile tube and add 1 &#181;g of each reporter plasmid DNA and mix gently by pipetting.</li>
	<li>Add 3 &#181;L of the transfection reagent to the DNA mixture, mix gently by pipetting and incubate for 15 min. Pre warm the transfection reagent to the ambient temperature of 22 &#176;C and vortex gently before use.</li>
	<li>Add the mixture drop-wise to the wells and gently distribute to the well.</li>
	<li>After 4 h, replace the supernatant with fresh medium containing the testing agents and solvent control. Incubate at 37 &#176;C until analysis. In this example, the mTOR inhibitors INK128 (100 ng/mL) and rapamycin (100 ng/mL) were used.</li>
</ol>
5. Fluorescence microscopy and flow cytometry
<ol>
	<li>Check cells for the red and green fluorescence under the fluorescence microscope (Figure 4). 
	NOTE: Red and green fluorescence correspond to the early and late BKPyV gene expression, respectively.</li>
	<li>After 72 h post transfection, aspirate the supernatant and wash the cells twice with 1 mL of cold PBS.</li>
	<li>Gently add 500 &#181;L of trypsin and turn the plate slightly to avoid premature detachment of the cells. This step is important to avoid cell doublets.</li>
	<li>After addition, remove the trypsin directly with the same pipette tip.</li>
	<li>Incubate the cells for at least 5 min at 37 &#176;C.</li>
	<li>Resuspend trypsinized cells with 1 mL of PBS containing 3% FCS and transfer the suspension in pre-labeled FACS tubes.</li>
	<li>Add DAPI (1 &#181;g/mL) prior to the FACS analysis.</li>
	<li>Analyze the cells using a flow cytometer. For each sample, measure at least 10,000 living cells, which are negative for DAPI staining.</li>
</ol>
6. Data analysis
<ol>
	<li>Import the data to the flow cytometry analysis software and add samples by Click and Drag into the workspace.</li>
	<li>Double click on the imported file and create a graph plot. Choose FSC-A versus SSC-A by clicking on the x- and y-axis. Gate the main cell population by clicking on Polygon at the toolbar and framing the cell population (Figure 5). Name the chosen cell population as required (for example &#34;single cells&#34;). The &#34;single cells&#34; will appear as a new workspace.</li>
	<li>Proceed with gating &#34;single cells&#34; for DAPI negative (i.e., &#34;living cells&#34;). To identify living cells compare the cell population with and without DAPI staining. In both plots choose FSC-A versus pacific-blue-A.</li>
	<li>Click on the rectangle and choose the DAPI negative cell population, name the chosen cell population &#34;living cells&#34;, and create a new dot-plot showing FITC (eGFP) versus PE (tdTomato) as described before.</li>
	<li>Add quadrants in &#34;living cells&#34; by choosing Quad from the toolbar. Gate the population by dragging the center of the quadrant to the edge of each population. The quadrants represent 1) eGFP-tdTOM-, 2) eGFP-tdTOM+, 3) eGFP+tdTOM-, 4) eGFP+tdTOM+. 
	NOTE: Due to the spectral overlay of emission spectra between FITC and PE, initial compensation is essential to distinguish between red and green signals and to achieve accurate results.</li>
	<li>Determine mean fluorescence intensities (MFIs) by right clicking on the quadrant and choosing Add Statistics. Choose Mean and click OK. The mean MFI is now displayed below the population in the section &#34;statistics&#34;. Quadrants 2 and 4 correspond to early expression, while quadrants 3 and 4 represent late expression.</li>
	<li>Add additional replicates in the same manner for statistical power.</li>
	<li>For data interpretation plot MFI values of early and late into a bar plot:</li>
	<li>To compare NCCR activities obtained from different donors or virus strains, add an archetypical control or the Dunlop strain16 and set its relative MFIs to 100%, and calculate the relative MFI values. Repeat with each replicate and determine mean and standard deviation.</li>
	<li>To evaluate an effect of potential compounds on the transcriptional activity, set the solvent-control to 100% and plot the MFI of the treated cells.</li>
</ol>

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Johannes Korth has received grants for speaker&#39;s fee and travel expenses from Astellas and Novartis. Marek Widera has received consultancy fees from Novartis. Oliver Witzke has received grants for clinical studies, speaker&#39;s fee, honoraria and travel expenses from Amgen, Alexion, Astellas, Basilea, Biotest, Bristol-Myers-Squibb, Correvio, Chiesi, Gilead, Hexal, Janssen, Dr. F. K&#246;hler Chemie, MSD, Novartis, Roche, Pfizer, Sanofi and TEVA.

In this article, a commonly used method is presented that allows for the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity. The NCCR activity can be measured simultaneously and does not need lysis of the transfected cells. Furthermore, a relatively large number of cells can be analyzed and the co-transfection of additional markers for normalization of the fluorescence values is not necessary.
A critical part of this method is that the cloned NCCR sh...

Polyomaviruses represent an independent family of small double-stranded DNA (dsDNA) viruses with the Simian virus 40 (SV40) as a prototype species. The primary infection mainly occurs during the childhood, which usually proceed without disease symptoms and usually cause latent infections in immune competent hosts. The BK-polyomavirus (BKPyV) mainly persists in renal tubules cells without causing nephropathologies, however, in case of impairment of the immune-competence after renal transplantation the virus can reactivate and cause severe damages and impaired graft function when reaching a high viremia (1 x 104 BKPyV DNA copies/mL)1,2. In approximately 10% of kidney transplant recipients, reactivation of the BK-polyomavirus (BKPyV) results in a polyomavirus associated nephropathy (PyVAN), which was up to 80% associated with a high risk of renal allograft failures3,4. Since no approved antiviral agents are available, current therapy is based on the reduction of immunosuppression. Interestingly, mTOR inhibitors seem to have an antiviral effect; thus, switching the immunosuppressive therapy to mTOR-based immunosuppression might represent an alternative approach to prevent progression of the BKPyV viremia5,6,7. However, the mTOR-based antiviral mechanism is currently still incompletely understood. Thus, methods measuring the impact of potential antiviral agents in clinically relevant concentrations are required.
The circular genome of the BKPyV consists of approximately 5 kb harboring a non-coding control region (NCCR) that serves as an origin of replication and concomitantly a bidirectional promoter driving the expression of early and late phase mRNA transcripts. Since spontaneously occurring NCCR-rearrangements, deletions, and duplications are found in pathogenic BKPyV8 and significantly accumulated in patients suffering from PVAN5,9, a comparison of archetypical (wt) and re-arranged (rr) NCCR-activities are helpful to characterize viral replicative fitness.
As summarized in Figure 1, this protocol describes a commonly used method to measure BKPyV NCCR transcriptional activity by quantifying the fluorescence of two fluorophores tdTomato and eGFP expressed from a reporter plasmid5,9,10,11. The procedure is performed in the presence of the SV40 large T antigen (lTAg), which allows to analyze the impact of potential antiviral agents on the early and late NCCR-activity separately5. This assay further analyzes the impact of rearrangements on the NCCR activity and comparison with wt-NCCRs5,9. The reporter plasmid harbors the SV40 late polyadenylation signal downstream of each fluorophore open reading frame to ensure comparable and efficient processing of both transcripts for tdTomato and eGFP, respectively. Compared to qRT-PCR based methods5,12, this FACS-based approach represents a low cost and high throughput compatible alternative since no complicated extraction protocols for infected cell culture and no expensive antibodies for immune fluorescence staining are needed. Furthermore, since a defined amount of fluorescent cells are analyzed via flow cytometry, the analysis of cell cycle inhibiting agents is also possible in a quantitative manner.

<table border="0" cellpadding="0" cellspacing="0" style="width:959px;" width="957">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">100 bp ladder</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">N3231</td>
			<td style="width:379px;">Any ladder with a range up to 1 kb can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">2-log ladder</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">N0550</td>
			<td>Any ladder with a range up to 10 kb can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Agar-Agar, Kobe I</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">5210.3</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">AgeI-HF</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">R3552</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Ampicillin Natriumsalz Cellpure</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">HP62.1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Aqua ad iniectabilia</td>
			<td style="width:127px;">Bbraun</td>
			<td style="width:151px;">2351744</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">BD FACSCanto&#8482; II</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">BD FACSDiva</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DAPI</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:151px;">10236276001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DFC450C camera module</td>
			<td style="width:127px;">Leica</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any camera can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DMEM</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">41966-029</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DMIL LED microscope</td>
			<td style="width:127px;">Leica</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any fluorescence microscope can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DNA Blood Mini Kit</td>
			<td style="width:127px;">Qiagen</td>
			<td style="width:151px;">51104</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">E.coli DH5alpha Competent Cells</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:151px;">18258012</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">FBS Superior</td>
			<td style="width:127px;">MerckMillipore</td>
			<td style="width:151px;">50615</td>
			<td style="width:379px;">Any FBS can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">FlowJo v10.5.3</td>
			<td style="width:127px;">FlowJo, LLC</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any flow cytometry software FBS can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Gel Loading Dye, Purple (6X)&#160;</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">B7024</td>
			<td>Any 6x loading dye can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HEK293T cells</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:151px;">11268</td>
			<td>These cells constitutively express the simian virus 40 (SV40) large T antigen, and clone 17 was selected specifically for its high transfectability.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HERAcell&#174; 240i CO2 Incubator</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:151px;"></td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HotStar PCR kit</td>
			<td style="width:127px;">Qiagen</td>
			<td style="width:151px;">203203</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Intas Gel documentation system</td>
			<td style="width:127px;">Intas</td>
			<td style="width:151px;"></td>
			<td>Any visualisation system for stained DNA containing agarose gels can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Low Melt Agarose</td>
			<td style="width:127px;">Biozym</td>
			<td style="width:151px;">850081</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Opti-MEM</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:151px;">31985070</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">pBKV (34-2)</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:151px;">45025</td>
			<td>Plasmid harboring the full-length genome of BKPyV strain Dunlop; was used as a positive control; DNA Seq. Acc.: KP412983</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PBS</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR Cycler MJ Mini 48-Well Personal Cycler</td>
			<td style="width:127px;">Bio-Rad</td>
			<td style="width:151px;">discontinued product</td>
			<td style="width:379px;">Any thermocycler can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR Nucleotide Mix, 10 mM</td>
			<td style="width:127px;">Promega</td>
			<td style="width:151px;">#C1145</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR1 and PCR2 Primers</td>
			<td style="width:127px;">metabion</td>
			<td style="width:151px;">not applicable</td>
			<td>Desalted. Dilute to (10 &#181;M) with PCR grade water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PenStrep (100x)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">15140-122</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Roti&#174;-GelStain</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">3865</td>
			<td>A fluorescence based stain for measuring dsDNA concentration</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">SpeI-HF</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">R3133</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">T4-DNA Ligase</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">M0202</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">TransIT LT1</td>
			<td style="width:127px;">Mirus</td>
			<td style="width:151px;">MIR2300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Trypsin 0.05% - EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">25300-054</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">ZymoPURE II Plasmid Kit</td>
			<td style="width:127px;">Zymo</td>
			<td style="width:151px;">D4201</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
	</tbody>
</table>

measurement of bk-polyomavirus non-coding control region driven transcriptional activity via flow cytometry

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

In this representative experiment, the BK-polyomavirus Non-Coding Control Region driven transcriptional activity was measured via flow cytometry. In addition, a mTOR inhibitor, which might be used to treat patients after BKPyV reactivation, was tested for its inhibition of the viral early gene expression. To this end, a dual fluorescence-reporter assay was used as published previously5. The overall workflow scheme of the experimental setup is illustrated in <strong...

Watch this Scientific Journal Video about Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry at JoVE.com

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

measurement-bk-polyomavirus-non-coding-control-region-driven

Research

JoVE Journal

Genetics

8.9K Views.  University of Duisburg-Essen, University Hospital Essen. In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Video: Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

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.

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.

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