Department of Molecular Microbiology, Faculty of Medicine, Technion - Israel Institute of Technology
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Brestovitsky, A., Sharf, R., Kleinberger, T. Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death. J. Vis. Exp. (68), e4442, doi:10.3791/4442 (2012).
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.
1. Generation of Inducible Cell Lines
2. Induction of Knockdown and Transfection
3. DAPI Assay in Transfected Cells
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).
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.
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.
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.
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. Click here to view larger figure.
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.
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.
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 which may not be efficient in many cell lines, and over the use of viruses that require repeated preparation and sometimes an additional short-term selection. The cell lines, once generated, do not require additional preparation. Furthermore, in our hands, transient transfections of shRNA constructs were much less efficient in knocking down gene expression compared with induction of shRNA expression in the selected cell lines. It is recommended however to ascertain in each experiment that the drug-induced knockdown remained efficient. A potential disadvantage of the use of stable cell lines for conditional knockdown of gene expression is that they may have acquired additional changes during the selection process that could potentially affect the outcome of experiments. However, to overcome this problem, one can carry out the experiment in two parallel cell lines or reverse the knockdown effect by introduction of a plasmid expressing a shRNA-resistant WT protein. If the WT protein eliminates the effect of the shRNA, then a clonal effect can be ruled out.
Classical, caspase-dependent apoptosis, can be assayed by numerous methods, including detection of caspase activation, annexin-V staining, Tunnel staining, detection of a sub-G1 cell population by FACS, etc.10. However, E4orf4 induces a non-classical, caspase-independent cell death in many cell lines and several methods for apoptosis detection are not applicable for detection of this unique mode of cell death6,7,11,12. It has been previously shown that the most typical morphologies associated with E4orf4-induced cell death include membrane blebbing, nuclear condensation or fragmentation, and cell detachment 13. Therefore we usually measure E4orf4-induced cell death by assaying nuclear condensation and fragmentation, or cell loss.
When utilizing the DAPI assay, one should pay special attention to the following points: 1) To maintain optimal objectivity of the test, it is highly recommended that the assay will be performed in a "blind fashion" so that the person who counts the number of cells containing nuclei with apoptotic morphologies will not be aware of the sample identity. 2) It is recommended to maintain stringent criteria to identify nuclear apoptotic morphologies which should not be confused with mitotic nuclei or nuclei with uneven morphologies (see Figure 5). Additional cell death assays can also be employed as a read-out of the experiment, such as clonogenic cell survival assays8.
No conflicts of interest declared.
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.
|High glucose DMEM||Gibco||41965|
|Tet system approved FBS||Clontech||631106|
|paraformaldehyde||Electron Microscopy Sciences||15710|