This work was supported (in part) by THE ISRAEL SCIENCE FOUNDATION (Grant 399/11), by the Deutsche Forschungsgemeinschaft (DFG) within the framework of The German-Israeli Project Cooperation (DIP), and by the Rappaport Family Institute for Research in the Medical Sciences.

1. Generation of Inducible Cell Lines
<ol>
 <li>Prior to the experiment one needs to find the minimal concentration of the drug G418 which kills all the cells used for preparation of the desired cell lines. For this purpose, plate the cells of choice in several duplicate plates at 70% confluency in the medium that will be used during the experiment and add increasing concentrations of G418 (0-1,000 &mu;g per ml). Monitor the cells daily to determine the minimal G418 concentration that kills the cells efficiently. Cell death is observed within 3-7 days.</li>
 <li>Prior to generation of cell lines it is recommended to verify by transient transfection assays that the plasmid expressing the shRNA can reduce to some extent expression of the gene under study. This may be done in any cell line that can be efficiently transfected.</li>
 <li>Plate T-REx-293 cells at a density of around 5x106 cells per 10 cm plate in 8 ml of DMEM medium (containing 10% Tet system approved FBS, 2mM L-glutamine, 100 units per ml penicillin and 0.1 mg per ml streptomycin, 5 &mu;g per ml blasticidin). Incubate the plates overnight at 37 &deg;C, 5% CO2.</li>
 <li>On the following day, change the medium to 8 ml fresh medium prior to transfection.</li>
 <li>Transfect the cells using 10 &mu;g of pSuperior.neo+GFP plasmid (Figure 1) encoding Acf1 or SNF2h shRNA driven by a tetracycline-inducible H1 promoter, as well as the neomycin-resistance gene fused to GFP and driven by a constitutive PGK promoter. Add the DNA to 500 &mu;l of 150 mM NaCl. Add the jetPIE reagent to another aliquot of 500 &mu;l of 150 mM NaCl at 2 &mu;l per &mu;g DNA. Add the jetPIE solution to the DNA and mix well by vortexing. Incubate for 15 min at room temperature. Gently pipette DNA complexes onto the 10 cm plates containing 70-80% confluent cells. Return the plates to the incubator.</li>
 <li>The next day replace the medium with a selective medium (DMEM medium as described above containing an additional 500 &mu;g per ml G418).</li>
 <li>For the next two weeks monitor the cells and replace the selective medium with a similar fresh medium every 3-4 days until colonies appear and can be visualized without a microscope.</li>
 <li>Prior to isolation of colonies, prepare 24-well plates with selective medium.</li>
 <li>Identify colonies by eye, and mark their location at the bottom of the plate using a colored marker. Verify under the microscope that the colonies are well separated.</li>
 <li>In the sterile hood, aspirate the medium from the plate, gently rinse with warm PBS and aspirate well all remaining liquid. Add 3 &mu;l of 0.25 % trypsin/EDTA to one colony on the plate. Pipette the trypsin repeatedly until the cells detach and add them to one of the wells in the 24-well plate. Repeat this for several other colonies on the plate.</li>
 <li>Grow the cells at 37 &deg;C, 5% CO2 until they fill the well. Then split the cells derived from each of the original colonies into three wells, each in a separate 12-well plate, and incubate overnight.</li>
 <li>On the following day, replace the medium in one plate with medium containing 1 &mu;g per ml doxycycline from a stock solution prepared in double-distilled water (1-5 mg per ml). Replace the medium in a control plate with medium without doxycycline. Incubate the cells for 72 hr at 37 &deg;C, 5% CO2.</li>
 <li>Harvest cells from one treated and one untreated well of each cell clone for preparation of protein extracts and Western blot analysis to determine the efficiency of Acf1 or SNF2h knockdown. Use antibodies to Acf1 or SNF2h as well as antibodies to alpha-Tubulin, serving as a loading control. Use the cells in the third well, left untreated, for expansion of selected cell clones in which doxycycline addition led to at least 50% reduction in the level of the protein under study. Freeze aliquots of these cells in 90% FBS, 10% DMSO for further use.</li>
</ol>
2. Induction of Knockdown and Transfection
<ol>
 <li>Plate cells in the selective medium in 10 cm plates. Add doxycycline at 1 &mu;g per ml to half the plates and incubate at 37&deg;C, 5% CO2 for 48-72 hr (see Figure 4).</li>
 <li>After 48-72 hr, trypsinize the cells from each group of cells, count them, and plate 1.5x106 cells per 6 cm plate in the same medium, with or without doxycycline. Incubate the cells at 37&deg;C, 5% CO2 overnight. Plan so that each group of plates from section 2.1 will provide the cells for twelve 6 cm plates, including two duplicates for the DAPI assay for each point as well as one plate for Western blot analysis of each sample (see Figure 4).</li>
 <li>On the following day transfect the cells in 6 cm plates with the following combinations of plasmids: 1 &mu;g of an empty plasmid or an identical quantity of the plasmid encoding E4orf4, together with 4 &mu;g of a vector expressing Acf1-GFP or SNF2h-GFP rendered resistant to the shRNA in the cell clones by introduction of silent mutations, or 3 &mu;g of the corresponding empty vector and 1 &mu;g plasmid expressing GFP (Figure 4). Use the jetPIE reagent as described above (10 &mu;l per 5 &mu;g DNA) to prepare the transfection mix. For each sample, prepare a transfection mix for three plates.</li>
 <li>Following a 15 min incubation of DNA with the jetPIE reagent at room temperature, gently pipette equal amounts of DNA complexes from each transfection mix onto three 6 cm plates and incubate at 37&deg;C, 5% CO2 overnight.</li>
</ol>
3. DAPI Assay in Transfected Cells
<ol>
 <li>The next day, extract proteins from one plate per sample for Western blot analysis to determine that Acf1 or SNF2h have been efficiently knocked down and that E4orf4 was expressed equally in the different samples.</li>
 <li>Aspirate the medium from the plates intended for the DAPI assay, wash the plates gently with PBS and add 1 ml of 4% paraformaldehyde prepared in PBS to cover the cells. Preparation of the paraformaldehyde solution and treatment of the cells with this chemical should be carried out in a chemical hood. Incubate for 15 min at room temperature without shaking.</li>
 <li>Aspirate the paraformaldehyde and wash three times with PBS, rocking at room temperature for 5 min each time. Add 80% ethanol kept at -20 &deg;C and incubate at -20 &deg;C for at least 1 hr. The plates can be kept under ethanol at -20 &deg;C for several days as long as they are sealed well to prevent ethanol evaporation and drying.</li>
 <li>Aspirate the ethanol and wash the cells twice with PBS and once with PBS-BT (PBS containing 0.5% BSA and 0.05% tween-20), rocking at room temperature for 5 min each time.</li>
 <li>To prevent non-specific antibody binding, block the cells in 1 ml PBS-BT buffer containing 10% goat serum for 20 min while rocking at room temperature. Then wash the cells twice in PBS-BT, 5 min each wash.</li>
 <li>Incubate the cells with the primary antibody (an E4orf4-specific antibody in our case) in 1 ml PBS-BT for 1 hr while rocking at room temperature. Then wash twice in PBS-BT and once in PBS containing 0.1% BSA, 5 min each wash.</li>
 <li>Incubate the cells in 1 ml of PBS-0.1% BSA containing the appropriate secondary fluorescently-labeled antibody and 0.5 &mu;g per ml final concentration of DAPI for 40 min while rocking at room temperature in the dark. Then wash with PBS for 5 min, dry well by aspiration and keep the plates upside down for an additional hour to overnight to achieve complete drying. Then mount cover slides on the cells, using Fluoromount-G solution. Keep plates at 4 &deg;C in the dark until ready to count the apoptotic nuclei.</li>
 <li>Ask a colleague to identify the various plates by new numbers, so counting apoptotic nuclei in the various samples can be done in an unbiased way.</li>
 <li>Visualize the cells under an upright fluorescent microscope at a magnification of 400X (in air) or 630X (in immersion oil). Identify transfected cells which are stained for the various proteins expressed in each sample, and count the number of condensed or fragmented nuclei visualized by DAPI staining within the transfected cell population. Monitor several fields and count a total of at least 200 transfected cells.</li>
 <li>Calculate the percentage of nuclei with apoptotic morphology within the transfected cell population. Repeat the experiment, with duplicates, 2-3 times to calculate the statistical significance of changes between the various samples.</li>
</ol>
4. Representative Results
The efficiency of obtaining colonies in which conditional knockdown of gene expression has been successful is variable, depending on the gene involved. In our hands, we obtained 7 successful colonies out of 12 tested for Acf1 and 6 out of 18 for SNF2h. Figure 2 shows a representative plate of selected cell clones (Figure 2A) as well as a single colony (Figure 2B). Figure 3 shows a representative blot prepared with proteins extracted from cells of various clones grown in the presence or absence of doxycycline for 72 hr. The blot was stained with antibodies to SNF2h and to alpha-Tubulin, serving as a loading control. Two of the clones (number 4 and 5) showed a strong reduction in SNF2h levels upon doxycycline induction. It should be noted that although the pSuperior.neo+GFP plasmid (Figure 1) used for generation of the cell lines encodes a neo-GFP fusion protein, the green fluorescence in the stable cell lines was very low and expression of other GFP fusion proteins introduced transiently into these cells could be easily detected above the background green fluorescence.
A schematic representation of the experiments carried out in the cell clones to measure the effect of Acf1 or SNF2h knockdown on E4orf4-induced cell death is shown in Figure 4. The time required for knockdown of gene expression by doxycycline treatment can vary depending on the stability of the protein whose levels should be reduced. For Acf1 and SNF2h, a 72 hr treatment was required for efficient knockdown at the protein level.
Figure 5 shows an example of cells expressing E4orf4 and GFP and undergoing cell death manifested by the appearance of DAPI-stained nuclei with a condensed or fragmented morphology. Figure 6 shows the results of a representative experiment demonstrating that doxycycline-induced Acf1 knockdown led to an increase in E4orf4-stimulated cell death (Figure 6A). This increase did not result from an increase in E4orf4 levels (Figure 6B). Furthermore, restoration of Acf1 to the doxycycline-induced cells diminished the increase in E4orf4 toxicity to the levels observed in uninduced cells (Figure 6).
<img alt="Figure 1" src="/files/ftp_upload/4442/4442fig1.jpg" fo:content-width="5.5in" fo:src="/files/ftp_upload/4442/4442fig1highres.jpg"/> 
Figure 1. Map of the pSuperior.neo+GFP plasmid. The map shows the tetracycline-inducible H1 promoter driving shRNA expression and the PGK promoter driving expression of a neo-GFP fusion protein. The map was adapted from the OligoEngine pSuperior manual.
<img alt="Figure 2" src="/files/ftp_upload/4442/4442fig2.jpg" /> 
Figure 2. Identification of neomycin-resistant colonies. Images of a plate containing colonies (A) and of a single colony (B) are shown. The colonies were obtained by culturing the cells for 14 days in a selective medium containing neomycin.
<img alt="Figure 3" src="/files/ftp_upload/4442/4442fig3.jpg" /> 
Figure 3. Examination of knockdown efficiency in selected colonies. Cells of several colonies selected in the presence of neomycin were plated in duplicate wells. One well of each sample was induced by doxycycline (+) and one well was left untreated (-). Proteins were extracted 72 hr post-induction and chromatographed on SDS-PAGE. The Western blot was stained successively with antibodies to SNF2h and to alpha-Tubulin (serving as a loading control) and shows SNF2h levels in induced and control cells.
<img alt="Figure 4" src="/files/ftp_upload/4442/4442fig4.jpg" /> 
Figure 4. Planning a typical knockdown-DAPI assay experiment. Successive steps of the experiment are shown, including doxycycline treatment to achieve Acf1 or SNF2h knockdown, a transfection to restore Acf1 or SNF2h expression or to introduce an empty vector and to introduce E4orf4 or its corresponding empty vector into the cells, and analysis of the results. Doxycycline-treated plates are shown in red (Dox) and control plates are marked in blue. <a href="https://www.jove.com/files/ftp_upload/4442/4442fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/4442/4442fig5.jpg" /> 
Figure 5. Detection of E4orf4-induced cell death by the DAPI assay. Cells which underwent the various treatments described in Figure 4 were fixed and stained with E4orf4-specific antibodies and DAPI to visualize the nuclei of transfected cells. This specific picture was taken from a sample containing E4orf4 and the control GFP protein. (A) GFP. (B) E4orf4. (C) DAPI. (D) Merged images. The white arrows mark GFP- and E4orf4-transfected cells containing nuclei with apoptotic morphologies. Red arrows mark nuclei with irregular shapes which are not counted as apoptotic nuclei. Asterisks mark mitotic nuclei or nuclei that have just divided.
<img alt="Figure 6" src="/files/ftp_upload/4442/4442fig6.jpg" /> 
Figure 6. Acf1 knockdown enhances E4orf4-induced cell death. (A) Cells from the T-REx-293- derived cell line expressing Acf1-shRNA from a tetracycline-inducible promoter were induced with doxycycline (+Dox) or left untreated (-Dox). Three days later, the cells were transfected with plasmids expressing E4orf4 (+E4orf4) or an empty vector (-E4orf4) together with an empty vector or a plasmid expressing GFP-tagged Acf1, which was rendered resistant to the shRNA by the introduction of silent mutations. The cells were fixed twenty-four hours after transfection and stained with antibodies to E4orf4 and with DAPI. Induction of cell death was measured by the DAPI assay described above and the percentage of transfected cells with condensed or fragmented nuclei was determined. A representative experiment with three replicates is shown, and error bars represent the standard deviation. (B) Cells from parallel plates were harvested for Western blot analysis. The blot was stained with antibodies to E4orf4, GFP, and Acf1. The endogenous Acf1 and Acf1-GFP are shown in two separate panels.

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	<li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science (New York, N.Y). 296, 550-553 (2002).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+suppression+of+tumorigenicity+by+virus-mediated+RNA+interference.">Stable suppression of tumorigenicity by virus-mediated RNA interference.</a> Cancer. 2, 243-247 (2002).</li><li>Czauderna, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+shRNA+expression+for+application+in+a+prostate+cancer+mouse+model.">Inducible shRNA expression for application in a prostate cancer mouse model.</a> Nucleic acids research. 31, e127 (2003).</li><li>Matsukura, S., Jones, P. A., Takai, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+conditional+vectors+for+hairpin+siRNA+knockdowns.">Establishment of conditional vectors for hairpin siRNA knockdowns.</a> Nucleic acids research. 31, e77 (2003).</li><li>Wiznerowicz, M., Trono, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+suppression+of+cellular+genes:+lentivirus+vector-mediated+drug-inducible+RNA+interference.">Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference.</a> Journal of. 77, 8957-8961 (2003).</li><li>Brestovitsky, A., Sharf, R., Mittelman, K., Kleinberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adenovirus+E4orf4+protein+targets+PP2A+to+the+ACF+chromatin-remodeling+factor+and+induces+cell+death+through+regulation+of+SNF2h-containing+complexes.">The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes.</a> Nucleic acids research. 39, 6414-6427 (2011).</li><li>Livne, A., Shtrichman, R., Kleinberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+activation+by+adenovirus+E4orf4+protein+is+cell+line-specific+and+is+mediated+by+the+death+receptor+pathway.">Caspase activation by adenovirus E4orf4 protein is cell line-specific and is mediated by the death receptor pathway.</a> J. 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No conflicts of interest declared.

Knockdown of specific gene expression is an important approach to the investigation of the contribution of a protein to regulatory pathways. Since constitutive knockdown of expression of essential genes negatively affects cell proliferation, their study may require either transient introduction of siRNAs or application of stable conditional knockdown systems. Generation of stable cell lines with the capacity for drug-induced expression of shRNAs described here, provides an advantage over transient transfections wh...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>High glucose DMEM</td>
 <td>Gibco</td>
 <td>41965</td>
 <td></td>
 </tr>
 <tr>
 <td>Tet system approved FBS</td>
 <td>Clontech</td>
 <td>631106</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen Strep</td>
 <td>Gibco</td>
 <td>15140</td>
 <td></td>
 </tr>
 <tr>
 <td>0.25% Trypsin-EDTA</td>
 <td>Gibco</td>
 <td>25200</td>
 <td></td>
 </tr>
 <tr>
 <td>T-REx-293 cells </td>
 <td>Invitrogen</td>
 <td>R710-07 </td>
 <td></td>
 </tr>
 <tr>
 <td>G418</td>
 <td>Sigma</td>
 <td>A1720</td>
 <td></td>
 </tr>
 <tr>
 <td>blasticidin</td>
 <td>Invitrogen</td>
 <td>R210-01</td>
 <td></td>
 </tr>
 <tr>
 <td>pSuperior.neo+GFP plasmid</td>
 <td>OligoEngine</td>
 <td>VEC-PBS-0007/0008</td>
 <td></td>
 </tr>
 <tr>
 <td>jetPIE</td>
 <td>Polyplus transfection</td>
 <td>101-40</td>
 <td></td>
 </tr>
 <tr>
 <td>doxycycline</td>
 <td>Sigma</td>
 <td>D9891</td>
 <td></td>
 </tr>
 <tr>
 <td>paraformaldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>15710</td>
 <td></td>
 </tr>
 <tr>
 <td>4',6-diamidino-2-phenylindole (DAPI)</td>
 <td>Sigma</td>
 <td>D9542</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluoromount-G</td>
 <td>SouthernBiotech</td>
 <td>0100-01</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of cell-lines for conditional knockdown of gene expression and measurement of the knockdown effects on e4orf4-induced cell death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Watch this Scientific Journal Video about Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death at JoVE.com

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

preparation-cell-lines-for-conditional-knockdown-gene-expression

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10.9K Views.  Technion - Israel Institute of Technology. Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Video: Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

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

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

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

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

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

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

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

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

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

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

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

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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

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

In vitro Reconstitution of the Active T. castaneum Telomerase

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

In vitro Reconstitution of the Active T. castaneum Telomerase

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

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

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

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 ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...