Gene Silencing by RNAi in Mammalian Cells

UNIT 26.2

Frida Ponthan,1 Narazah Mohd Yusoff,2 Natalia Martinez Soria,1 Olaf Heidenreich,1 and Kelly Coffey1 1

Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom 2 Advanced Medical & Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia

This unit provides information how to use short interfering RNA (siRNA) for sequence-specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell C 2015 by John Wiley & Sons, transfection and transduction, are presented.  Inc. Keywords: RNA interference r siRNA r shRNA r transfection r electroporation r lentiviral transduction

How to cite this article: Ponthan, F., Yusoff, N.M., Soria, N.M., Heidenreich, O., and Coffey, K. 2015. Gene silencing by RNAi in mammalian cells. Curr. Protoc. Mol. Biol. 111:26.2.1-26.2.17. doi: 10.1002/0471142727.mb2602s111

INTRODUCTION For many years, specific and efficient modulation of mammalian gene expression was a very difficult and labor-intensive task. Particularly promising technologies for controlling gene expression were antisense oligonucleotides and ribozymes. In the case of antisense technology, there are a number of examples in which reasonably strong down-regulation of specific target genes was obtained. In most cases, however, a tedious and costly process of target-sequence optimization was necessary to obtain satisfactory results. The discovery of gene-specific silencing in mammalian cells mediated by RNA interference allows researchers to circumvent many problems of the traditional antisense protocols. RNA interference works in most cell types, and target sequence identification is straightforward. In most cases, by paying attention to a few rules of oligoribonucleotide design, it is sufficient to design three to five different siRNA molecules for each target gene. The induction of siRNA-mediated RNA interference (RNAi) in mammalian cells can be achieved by microinjection of siRNA, transfection of siRNA using cationic lipids, electroporation of siRNA, transfection of plasmids containing siRNA expression cassettes, or infection of cells by recombinant viruses such as lentiviruses that encode the siRNA gene of interest. Because determining an efficient cell transfection procedure for a particular cell line can be a rate-limiting step, this unit contains a method for siRNA transfection using a cationic lipid (see Basic Protocol), an alternative method involving electroporating siRNA (see Alternate Protocol 1), and a protocol for the use of lentiviral vectors for shRNA delivery (Alternate Protocol 2). In addition, procedures for annealing siRNA strands (see Support Protocol 1) and for production of lentiviral particles (see Gene Silencing Current Protocols in Molecular Biology 26.2.1-26.2.17, July 2015 Published online July 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142727.mb2602s111 C 2015 John Wiley & Sons, Inc. Copyright 

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Support Protocol 2) are also provided. Besides the specific experimental siRNAs, all experiments have to include at least one control siRNA unrelated to the target mRNA to evaluate nonspecific or toxic effects. NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. NOTE: All solutions and materials coming into contact with RNA must be RNase-free, and proper techniques should be used accordingly (see UNIT 4.1; Gilman, 2002). BASIC PROTOCOL

LIPOSOME-MEDIATED FORWARD AND REVERSE TRANSFECTION OF MAMMALIAN CELLS WITH siRNA Liposome-mediated transfection of mammalian cells with siRNA shares some similarity with DNA transfection protocols [see UNIT 9.4 (Hawley-Nelson et al., 2008) for additional information]. Similar considerations apply for siRNA and DNA transfection protocols. However, special transfection reagents have been developed to achieve optimal transfection efficiencies with siRNA. For some cell lines, specific DNA transfection reagents such as Lipofectamine PLUS (Invitrogen) appear to work the best. In addition, the user can determine whether to use a forward transfection procedure, where the siRNA-liposome complexes are added to adherent cells, or a reverse transfection procedure where the siRNA-liposome complexes are put into the plate first followed by the addition of cells. Further information regarding these options is given below.

Materials Mammalian cells to be transfected (e.g., LNCaP) Complete medium for 293 cells (see recipe) 20 μM annealed siRNA (see Support Protocol 1) Serum-free RPMI medium Lipofectamine RNAiMAX (Life Technologies) 6-well culture dishes 1.5-ml polypropylene tubes Centrifuge Additional reagents and equipment for mammalian cell tissue culture, including trypsinization (APPENDIX 3F; Phelan, 2006), northern blot hybridization (UNIT 4.9; Brown et al., 2004), RNase protection assay (UNIT 4.7; Gilman, 1993), RT-PCR (UNIT 15.8; Bookout et al., 2006), and immunoblotting (UNIT 10.8; Gallagher et al., 2008) NOTE: Lipofectamine RNAiMAX is used in this protocol, as it works well for LNCaP cells. Other transfection reagents can also be used by following manufacturer’s instructions. Lipofectamine RNAiMAX can be used to perform both forward and reverse transfections depending on user preference or requirements. For example, for large-scale experiments, reverse transfection is preferred over forward transfection, as it is less labor intensive. Similarly, if the cell line is difficult to transfect, this method may improve the transfection efficiency.

Perform forward transfection Gene Silencing by RNAi in Mammalian Cells

1a. Cultivate the mammalian cells to be transfected to late log phase in complete medium (APPENDIX 3F; Phelan, 2006).

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2a. Trypsinize and count cells (APPENDIX 3F; Phelan, 2006). Transfer to appropriate tubes and centrifuge for 5 min at 300 × g, room temperature. Discard supernatant and wash once with complete medium. Resuspend in complete medium. 3a. Seed 2 ml containing 1 × 105 cells in each well of a 6-well tissue culture plate and incubate 48 hr. 4a. Replace the medium with 2 ml fresh medium 4 hr before the start of the transfection protocol and return to the incubator. 5a. For each well, mix 2.5 μl of 20 μM annealed siRNA with 47.5 μl serum-free medium in a 1.5-ml polypropylene tube. 6a. In a second 1.5-ml reaction tube, dilute 7.5 μl Lipofectamine RNAiMAX in 42.5 μl serum-free medium. Vortex for 15 sec. 7a. Add Lipofectamine RNAiMAX solution (step 6) to the tube containing the siRNA mixture (1:1 ratio) and mix gently. 8a. Incubate for 20 min at room temperature. 9a. Add all of the siRNA transfection mix (100 μl) from step 7 in a drop-wise manner. Using this protocol as written (i.e., 20 μM siRNA stock), the final concentration of siRNA is 25 nM. The optimum siRNA concentration varies depending on the cell line or target gene and is typically between 1 and 100 nM.

10a. Incubate 16 to 48 hr and analyze target RNA by northern blot (UNIT 4.9; Brown et al., 2004), RNase protection assay (UNIT 4.7; Gilman, 1993), or RT-PCR (UNIT 15.8; Bookout et al., 2006). Alternatively, determine resulting protein levels by immunoblot (UNIT 10.8; Gallagher et al., 2008). In most cases, a decrease in target RNA level is accompanied by simultaneous depletion of the corresponding protein in cell extracts. However, some proteins may have a halflife of many hours in vivo, and thus the time course of RNA and protein degradation are not the same. In those cases, the cells have to be harvested at different time points. For example, RNA preparation can be done after 48 hr, whereas protein extracts should be prepared after 72 or 96 hr.

Perform reverse transfection 1b. Cultivate the mammalian cells to be transfected to late log phase in complete medium (APPENDIX 3F; Phelan, 2006). 2b. For each well, mix 2.5 μl of 20 μM annealed siRNA with 47.5 μl serum-free medium in a 1.5-ml polypropylene tube. 3. In a second 1.5-ml reaction tube, dilute 7.5 μl Lipofectamine RNAiMAX in 42.5 μl serum-free medium. Vortex for 15 sec. 4b. Add RNAiMAX solution (step 2) to the tube containing the siRNA mixture (at a 1:1 ratio) and mix gently. Immediately add the mixture into a well of a 6-well plate. 5b. Incubate for 20 min at room temperature. 6b. During this incubation period, prepare cells for transfection as follows. Trypsinize and count cells (APPENDIX 3F; Phelan, 2006). Transfer to appropriate tubes and centrifuge for 5 min at 300 × g, room temperature. Discard supernatant and wash once with complete medium, centrifuging at the same speed after the wash. Remove supernatant and resuspend in complete medium. Gene Silencing

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7b. Upon completion of step 5b, add 2.5 × 105 LNCaP cells in 1.9 ml of complete medium to the well containing the siRNA/RNAiMAX mixture. Mix gently. 8b. Incubate 16 to 48 hr and analyze RNA by Northern blot (UNIT 4.9; Brown et al., 2004), RNase protection assay (UNIT 4.7; Gilman, 1993), or RT-PCR (UNIT 15.8; Bookout et al., 2006). Alternatively, determine resulting protein levels by immunoblot (UNIT 10.8; Gallagher et al., 2008). ALTERNATE PROTOCOL 1

ELECTROPORATION OF MAMMALIAN CELLS WITH siRNA Many mammalian cell types can be easily and efficiently electroporated under very mild conditions resulting in cell survival rates >90%. Particularly with suspension cells, siRNA delivery by electroporation is superior to liposome-mediated transfection. Tests with fluorescently labeled siRNA and subsequent analysis by fluorescence-activated cell sorting indicate that up to 100% of the cells of the leukemic cell line Kasumi-1 can be transfected (Heidenreich et al., 2003). An additional advantage is that the electroporation protocol needs no particular transfection reagents and requires only a few steps. An increasing number of laboratories are now discovering the advantages of using electroporation for siRNA transfection instead of liposome-based methods (Bakshi et al., 2008; Ben-Ami et al., 2013; Wilkinson et al., 2013). In particular, suspension cells, such as Kasumi-1 or other leukemic cell lines, and also primary hematopoietic cell types, are good candidates for this method. These suspension cells seem to work well with electroporation and are often difficult to transfect with liposome-forming reagents. Since suspension cell lines tolerate this protocol very well, they are suitable for sequential electroporations, thereby overcoming limitations such as long protein half-life. For instance, leukemic cell lines such as Kasumi-1 or SEM can be sequentially electroporated every 3 to 4 days without substantial impact on cell viability (Ptasinska et al., 2012). Nonetheless, exceptions are observed among suspension cells, and there is no particular advantage in electroporating adherent cells.

Additional Materials (also see Basic Protocol) 4-mm electroporation cuvettes (e.g., Peqlab 71-2030) Square-wave electroporator (e.g., Fischer EPI 2500 (http://www.electroporation.eu) or BioRad Gene Pulser Xcell) Culture vessel Additional reagents and equipment for electroporation (UNIT 9.3; Potter and Heller, 2010) 1. Cultivate the mammalian cells to be transfected to late log phase in complete medium (APPENDIX 3F; Phelan, 2006). 2. Centrifuge cells 5 min at 300 × g, 21°C, and resuspend cell pellet in complete medium at a final concentration of 107 to 108 cells/ml. Alternatively, serum-free medium may be used. In that case, cells should be washed once with serum-free medium before resuspending them at 107 to 108 cells/ml.

3. Transfer 100 to 750 μl cell suspension into a 4-mm electroporation cuvette at room temperature. Add 20 μM annealed siRNA to a final concentration of 100 to 200 nM. Mix well by flicking the cuvette.

Gene Silencing by RNAi in Mammalian Cells

4. Place cuvette into the holder of a square-wave electroporator and electroporate at the desired voltage and time setting.

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For hematopoietic cell lines such as BAF3, K562, or Kasumi-1, settings of 300 to 350 V and 10 msec are recommended. To establish the electroporation conditions, the authors routinely electroporate 106 cells in 100 μl medium containing 200 nM of the corresponding siRNA for a constant time of 10 msec with varying voltage from 300 to 400 V. Some electroporation systems offer more variables (e.g., voltage, pulse time, resistance, impedance). The settings have to be determined for each cell line empirically. See UNIT 9.3 (Potter and Heller, 2010) for more detail.

5. Remove cuvette from holder and incubate 15 min at room temperature. 6. Return the electroporated cells to a culture vessel and dilute the cell suspension to the desired cell density. For many electroporated leukemic cell lines such as Kasumi-1 cells, a cell density of 5 × 105 /ml is appropriate. For hematopoietic suspension cells, transfection rates of up to 100% can be obtained with a fluorescently labeled siRNA; however, the authors observed rather poor transfection rates for fibroblast cell lines such as NIH3T3.

7. Analyze RNA by northern blot (UNIT 4.9; Brown et al., 2004), RNase protection assay (UNIT 4.7; Gilman, 1993), or RT-PCR (UNIT 15.8; Bookout et al., 2006). Alternatively, analyze target protein expression (UNIT 10.8; Gallagher et al., 2008). Decreases in RNA levels are visible within 8 hr after electroporation. Reduction of protein levels and the consequences of protein depletion depend on the half-life of the targeted protein. In the case of electroporated Kasumi-1 cells, the authors routinely observe 70% reduction of endogenous gene expression.

LENTIVIRAL TRANSDUCTION OF SUSPENSION CELLS USING SPINFECTION

ALTERNATE PROTOCOL 2

To establish stable expression of siRNA, cells can be transduced with lentiviral particles containing an shRNA expression vector. Using this technique, the shRNA cassette will be stably integrated into the genome, allowing for continuous or inducible siRNA expression. This method works for adherent cells as well, and can be particularly useful in an in vivo setting where a continued or induced siRNA expression “in situ” is an advantage.

Additional Materials (also see Basic Protocol) 8 g/liter Polybrene Phosphate-buffered saline (PBS; APPENDIX 2) Virus particles containing shRNA expression vector Appropriate selective antibiotic Doxycycline 24- and 48-well culture plates Centrifuge Additional reagents and equipment for flow cytometry (Robinson et al., 2015) Day 1 1. Cultivate the mammalian cells to be transduced to late log phase in complete medium (APPENDIX 3F; Phelan, 2006). 2. Spin 5 × 106 cells for 5 min at 300 × g, 21°C, and resuspend cell pellet in 5 ml complete medium. 3. Mix 5 ml of cell suspension (106 cells/ml) with 5 μl of 8 g/liter Polybrene. Polybrene (hexadimethrine bromide) is a cationic polymer used to increase the efficiency of infection of certain cells with a retrovirus in cell culture. It acts by neutralizing the charge repulsion between virions and sialic acid on the cell surface.

Gene Silencing

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4. Pipet 0.5 ml of this mix into 7 wells of a 48-well plate. Add PBS to the remaining wells to minimize evaporation. 5. Add 1, 2.5, 5, 10, 25, 50, and 100 μl of concentrated lentiviral supernatant to the 7 wells containing the cell culture/Polybrene mix, close with lid, and seal plate with Parafilm (for production of lentivirus, see Support Protocol 2). This step assumes that the virus titer has not been determined at that stage for the cell line to be transduced. The number of wells can be reduced if the virus titer is known.

6. Spin for 2 hr at 1,500 × g, 32°C. The centrifuge has to be preheated to 32°C.

7. Remove Parafilm and incubate cells overnight under standard culture conditions.

Day 2 8. Carefully remove 350 μl of supernatant from each of the 7 wells without disturbing the cells. 9. Add 850 μl of complete medium to each of the 7 wells, mix well, and transfer 800 μl to 7 wells in a 24-well plate. It is important to dilute the Polybrene the day after transduction to avoid toxic effects.

10. Put PBS in the remaining wells to avoid evaporation and incubate for 3 days.

Day 5 11. In the case of continuous shRNA expression being linked to expression of a fluorescent protein such as GFP or RFP, check fluorescent protein expression by flow cytometry (Robinson et al., 2015). 12. If the shRNA expression vector also contains an antibiotic resistance gene, start antibiotic selection for cells containing the shRNA expression vector. Before starting the selection, it is recommended to do a dose-response experiment to determine the concentration of the selective antibiotic of choice (e.g., puromycin, zeocin, blasticidin) that kills about 90% to 100% of untransduced cells after 3 days. This concentration is used for the selection of the transduced cells. For hematopoietic cell lines such as SEM and Kasumi-1, the concentration of puromycin is 0.5 to 1 μg/ml and 4 to 5 μg/ml for zeocin. Inducible shRNA expression vectors such as pTRIPZ contain a Tetracycline Response Element (TRE) upstream of the red fluorescent protein (RFP) and the shRNA cassette. Because they continuously express the Tetracycline Activator (TA), shRNA expression can be induced with doxycycline as in step 11.

11. Add doxycycline to a final concentration of 1 μg/ml to the remaining 200 μl of cell suspension in the 48-well plate. 12. Check for RFP expression by flow cytometry (Robinson et al., 2015) 72 to 96 hr after addition of doxycycline. To minimize the risk of multiple integrations, continue culturing the cells such that the virus concentration results in

Gene Silencing by RNAi in Mammalian Cells.

This unit provides information how to use short interfering RNA (siRNA) for sequence-specific gene silencing in mammalian cells. Several methods for s...
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