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. N., Nguyen, M., Marcellus, R. C., Branton, P. E., Shore, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E4orf4,+a+novel+adenovirus+death+factor+that+induces+p53-independent+apoptosis+by+a+pathway+that+is+not+inhibited+by+zVAD-fmk.">E4orf4, a novel adenovirus death factor that induces p53-independent apoptosis by a pathway that is not inhibited by zVAD-fmk.</a> J. Cell Biol. 140, 637-645 (1998).</li><li>Li, S. <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+induces+growth+arrest+and+mitotic+catastrophe+in+H1299+human+lung+carcinoma+cells.">The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells.</a> Oncogene. 28, 390-400 (2009).</li><li>Lavoie, J. N., Champagne, C., Gingras, M. -. C., Robert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+E4+open+reading+frame+4-induced+apoptosis+involves+dysregulation+of+Src+family+kinases.">Adenovirus E4 open reading frame 4-induced apoptosis involves dysregulation of Src family kinases.</a> J. Cell Biol. 150, 1037-1055 (2000).</li></ol>

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 ...

Research

JoVE Journal

Biology

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 ...

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

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 ...

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

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 ...

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

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 ...

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

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 ...

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

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 ...

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

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 ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...