Name: preparation of cell-lines for conditional knockdown of gene expression and measurement of the knockdown effects on e4orf4-induced cell death
Uploaded: 2012-10-21T14:00:00
Description: 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

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 ...

<ol>
	<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. Virol. 75, 789-798 (2001).</li><li>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=Adenovirus+type+5+E4+open+reading+frame+4+protein+induces+apoptosis+in+transformed+cells.">Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells.</a> J. Virol. 72, 2975-2982 (1998).</li><li>Shtrichman, R., Sharf, 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=Adenovirus+E4orf4+protein+interacts+with+both+Ba+and+B'+subunits+of+protein+phosphatase+2A,+but+E4orf4-induced+apoptosis+is+mediated+only+by+the+interaction+with+Ba.">Adenovirus E4orf4 protein interacts with both Ba and B' subunits of protein phosphatase 2A, but E4orf4-induced apoptosis is mediated only by the interaction with Ba.</a> Oncogene. 19, 3757-3765 (2000).</li><li>Galluzzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+cell+death+in+higher+eukaryotes.">Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.</a> Cell death and differentiation. 16, 1093-1107 (2009).</li><li>Lavoie, J. 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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 ...

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