Protocol

RNA Interference of Gene Expression (RNAi) in Cultured Drosophila Cells

Science's STKE  14 Aug 2001:
Vol. 2001, Issue 95, pp. pl1
DOI: 10.1126/stke.2001.95.pl1

Abstract

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

Introduction

The ablation of protein expression has long been the standard approach for deciphering the functions of a particular protein. The use of double-stranded RNA (dsRNA) to silence genes is possible in a variety of organisms including planarians, trypanosomes, zebrafish, Caenorhabditis elegans, Drosophila, mouse embryos, and most recently, mammalian cells (1-6). RNA interference of gene expression (RNAi) serves as a powerful tool for determining protein function. However, studies in the context of whole organisms are often too complicated to finely dissect the function of a protein in a cell. We therefore attempted to apply RNAi to cultured cells, because they constitute model systems in which signal transduction pathways and other cellular processes can be effectively dissected. The use of cultured Drosophila cells has several advantages. First, many Drosophila cell lines have been established and can be biochemically characterized for use in studying various cellular processes (7, 8). Second, many signal transduction pathways and other cellular processes have been highly conserved from Drosophila to mammals, making it possible to study complex biochemical problems in a genetically tractable model organism. Importantly, results obtained from the cell culture studies can be confirmed in the whole organism, because Drosophila is also amenable to RNAi analyses at the organismal level. Third, the use of dsRNA in Drosophila cell culture to silence expression of specific genes is technically simple, efficacious, and highly reproducible (9). The dsRNAs are efficiently internalized by the cells, thereby circumventing the problems generated by variable transfection efficiencies. Finally, the gene-silencing effect can be sustained through many cell divisions, as long as the protein of interest is not necessary for cell viability. Therefore, RNAi in Drosophila cell culture provides a highly effective method for determining the function of proteins identified in the Drosophila genome sequencing project, as well as for deciphering the functions of mammalian proteins that have Drosophila orthologues.

Materials

Drosophila S2 Cell Culture

75-cm2 tissue culture flasks

Drosophila cells (for example, S2) (American Type Culture Collection)

DES Serum-Free Expression Medium (Invitrogen)

Fetal bovine serum (Life Technologies)

Glutamine

Penicillin and streptomycin

Phosphate-buffered saline (PBS)

Schneider's Drosophila medium (Life Technologies)

Six-well tissue culture dishes

Trypan blue

Preparation of the Template DNA

Deoxyribonucleotide triphosphates (dNTPs): dATP, dCTP, dGTP, dTTP (0.2 mM each)

Diethylpyrocarbonate (DEPC)

DNA polymerase [for example, AmpliTAQ Gold polymerase (Applied Biosystems) or Vent polymerase] and appropriate 10× reaction buffer

High Pure PCR Product Purification kit (Roche Molecular Biochemicals)

Magnesium chloride

PCR primers (for further detail, see Instructions, Preparation of PCR Template for dsRNA Synthesis)

Preparation of the dsRNA

Agarose and other reagents needed for horizontal analytical gel

Autoclaved microcentrifuge tubes

Ethanol, 100%

MEGAscript T7 or T7-MEGAshortscript kit (Ambion)

Sodium acetate

Preparation of S2 Cell Extracts

2-mercaptoethanol (2-ME)

Benzamidine (Sigma-Aldrich)

Bromophenol blue

Cell Lifter (Costar)

Deoxycholate

Ethylenediaminetetraacetic acid (EDTA)

Glycerol

Leupeptin (Roche Molecular Biochemicals)

Needles, 25 gauge, 1.5 inch (3.8 cm) G 1 1/2

Nonidet P-40 or Igepal CA-630 (Sigma-Aldrich)

Note: Nonidet P-40 is no longer made, but according to the manufacturer, Igepal CA-630 is chemically indistinguishable from Nonidet P-40. Either detergent is suitable.

Pefablock (Roche Molecular Biochemicals)

Polyallomer ultracentrifuge microtube (Beckman #357448)

Sodium dodecyl sulfate (SDS)

Sodium fluoride

Sodium orthovanadate

Syringes, 1-cc

Trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) (Sigma-Aldrich)

Equipment

Hemacytometer

Mini-PROTEAN II electrophoresis and gel transfer system (Bio-Rad) or similar equipment for protein electrophoresis and blotting

Spectrophotometer

Tissue culture hood

TL-100 Ultracentrifuge (Beckman)

TLA-100.3 Rotor

Recipes

Recipe 1: S2 Cell Growth Media
Fetal bovine serum 10%
Penicillin 50 units/ml
Streptomycin 50 µg/ml
Add to Schneider's Drosophila medium.
Recipe 2: DEPC-Treated H20
Add DEPC to ddH2O at a final concentration of 0.2%. Stir overnight and autoclave to inactivate the remaining DEPC.
Note: Wear gloves and work in a fume hood when handling DEPC, because it is a suspected carcinogen.
Recipe 3: Sodium Acetate Solution
3M Sodium acetate, pH 5.2, prepared in DEPC-Treated H2O (Recipe 2).
Recipe 4: S2 RNAi Incubation Media
Glutamine 2 mM
Add to DES Serum-Free Expression Medium
Recipe 5: 200 mM Sodium Orthovanadate
1. Prepare a 200-mM-stock solution in water.
2. Adjust the pH to 10.0 using either 1N NaOH or 1N HCl. The starting pH of the sodium orthovanadate solution may vary. At pH 10.0 the solution will be yellow.
3. Boil the solution until it turns colorless (about 10 min).
4. Cool to room temperature.
5. Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0.
6. Store the activated sodium orthovanadate as 1-ml aliquots at -20°C.
Recipe 6: RIPA Lysis Buffer
NaCl 150 mM
Nonidet P-40 1%
Deoxycholate 0.5%
SDS 0.1%
Tris-HCl pH 8.0 50 mM
NaF 10 mM
EDTA pH 8.0 0.4 mM
Glycerol 10%
Store 500-ml aliquots of RIPA buffer at 4°C. At time of use, add sodium vanadate from 200 mM Sodium Orthovanadate stock (Recipe 5) to a final concentration of 1mM and add 100× PIC stock (Recipe 7) to a final concentration of 1×.
Recipe 7: Proteinase Inhibitor Cocktail (PIC), 100×
E-64 0.1 mM
Benzamidine 100 mM
Leupeptin 0.1 mM
Pefabloc 100 mM
Store 100-ul aliquots at -20°C for up to 2 months. Do not freeze and thaw an aliquot more than twice.
Recipe 8: Laemmli Loading Buffer (LLB), 5×
Tris-HCl pH 6.8 125 mM
Glycerol 10%
SDS 2%
2-ME 5%
Bromophenol blue 0.00125%

Instructions

Preparation of PCR Template for dsRNA Synthesis

If you do not have the cDNA of interest, the first step is to obtain it in the form of an expressed sequence tag (EST) from the Drosophila Genome Project (http://www.fruitfly.org/). From this EST sequence, PCR a template to be used for synthesizing your dsRNA. This PCR template is designed to represent a 300- to 1000-bp region of the EST containing 5′ T7 RNA polymerase binding sites (5′-TTAATACGACTCACTATAGGGAGA-3′) on each strand. The PCR template may include any part of the EST, either coding or 3′ or 5′ untranslated sequence. We have always used EST coding sequences to synthesize our PCR products. If you start with genomic DNA, make sure not to incorporate any intervening sequence into your PCR product.

Note: From this point on, care must be taken to use only RNase-free solutions and materials, and gloves should be worn to prevent RNase contamination.

1. Design two oligonucleotides, forward and reverse, as described above.

Note: To optimize your PCR reaction, your oligonucleotides should be designed for a melting temperature between 52°C and 58°C. Each C or G base pair contributes approximately 4°C, and each A or T base pair contributes approximately 2°C to the melting temperature.

2. Set up a typical 100-µl PCR reaction:

100 ng of EST DNA

150 ng of both forward and reverse oligonucleotides

Polymerase buffer, to final concentration of 1×

dNTPs, to final concentration of 0.2 mM each

5 units DNA polymerase

MgCl2, as required

H2O to a final volume of 100 µl

Note: We use either Vent or AmpliTAQ Gold polymerase. Determine empirically which DNA polymerase yields the better result for your particular PCR reaction.

3. Purify the completed PCR reaction with the High Pure PCR Product Purification column according to kit-supplied protocol.

4. Elute the PCR product from the column with 50 µl of DEPC-Treated H2O (Recipe 2).

Note: PCR templates must be free of RNases and inhibitors of the RNA synthesis reaction such as high salt, detergents, or EDTA.

5. Visualize the PCR product on an agarose gel to determine both its size and fidelity. The DNA must be at a concentration of 125 ng/ µl or greater to proceed to the next step.

Preparation of dsRNA

The Ambion MEGAscript T7 kit is used to synthesize dsRNA from templates > 0.5 kb, and the T7- MEGAShortscript kit is used for templates < 0.3 kb with the following modifications.

1. Thaw the reaction components, except the enzyme mix, at room temperature.

Note: Do not put on ice, because precipitates may form if reagents are chilled.

2. To a sterile 1.5-ml microcentrifuge tube at room temperature, add the following kit reagents in the order shown for a 20-µl reaction mix:

(8 - X) µl DEPC-Treated H2O (Recipe 2) (sufficient volume to make a 20-µl final reaction volume)

2 µl 10× Reaction Buffer

2 µl each of ATP, CTP, GTP, UTP mixes

X µl linearized DNA template (sufficient volume to deliver 1 µg of DNA)

2 µl enzyme mix

Note: Expected yields of dsRNA range from 0.1 to 0.4 mg. This reaction can be scaled up as desired to make large quantities of dsRNA.

3. Mix the tube gently by hand, then pulse the tube at maximum speed in a microcentrifuge to pellet liquid in the bottom of the tube.

4. Incubate at 37°C for 2 to 6 hours.

5. Precipitate reaction at -20°C with 3M Sodium Acetate Solution (Recipe 3) to a final concentration of 10% and 2.5 volumes of 100% ethanol.

6. Spin the samples for 15 min in a microcentrifuge at maximum speed.

7. Remove the ethanol and air dry the pellet for 15 min.

8. Resuspend the RNA pellet in about 50 µl of DEPC-Treated H2O (Recipe 2).

9. Anneal the RNA strands by heating to 65°C for 30 min and then slowly cooling to room temperature.

10. Determine the OD at 260 nm.

11. Dilute the sample to a final concentration of 3 µg/µl. This will be a 1× concentration dsRNA solution.

12. Visualize the dsRNA on a 1% agarose gel to check the integrity and size of the dsRNA (Fig. 1).

Fig. 1.

Representative examples of dsRNA preparations. Six µg of dsRNA were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining.

Note: Each of the preparations shown was effective at ablating protein expression (Fig. 1). Therefore, the presence of a few minor dsRNAs of varying size, or the presence of a smear, is of no concern.

13. Store the dsRNA at -20°C. Preparations of dsRNA appear to be stable for a minimum of 12 months at -20°C, with no loss of efficacy.

S2 Cell Culture

S2 cells are propagated in S2 Cell Growth Medium (Recipe 1), in 75-cm2 tissue culture flasks at 25°C. S2 cells grow both in suspension and attached to the flask. Passage the cells at a 1:10 dilution into a new flask once they achieve a cell density of approximately 6 × 106 cells/ml. The cells that are attached to the flask are easily dislodged by gently tapping the side of the flask with your hand and drawing the medium up and down with a pipet to thoroughly mix the cells in the flask. S2 cells grow well as long as they are not subcultured to a very low density.

Note: We have found that S2 cells are very particular regarding the FBS source. We use only Life Technologies FBS from a lot number that has been tested to ensure that the S2 cells will grow.

1. Mix the cells in a 75-cm2 tissue culture flask.

2. Remove 0.1 ml of cells and place in a 1.5-ml microcentrifuge tube.

3. Mix 80 µl of PBS with 10 µl of cells and 10 µl of Trypan blue.

4. Place 10 µl of the cell suspension on a hemacytometer.

5. Count the round, refractile live cells on both grids of the hemacytometer and calculate the concentration of cells. Before proceeding, ensure that > 95% of the cells are alive.

Note: When visualizing the cells under the microscope, those cells that are stained blue are dead. Count the live cells on both grids and take the average. The average of the number of cells counted multiplied by the dilution factor (in this case, 10) multiplied by 104 equals the number of cells/ml.

6. Pellet sufficient cells for all of the experimental conditions and for an untreated control by centrifugation at 500g for 10 min.

7. Resuspend the cells at a final concentration of 1 × 106 cells per ml in S2 RNAi Incubation Medium (Recipe 4).

8. Place 1 ml of cells per well into a 6-well cell culture dish.

Note: Be sure to plate 1 well of cells that will not be treated with dsRNA as your control.

9. Immediately add 15 µg of dsRNA (5 µl of 3 µg/µl stock) and mix plates by hand with a back-and-forth motion for 10 to 15 s to mix the dsRNA and cells.

10. Incubate at room temperature for 30 to 60 min.

11. Add 2 ml of S2 Cell Growth Media (Recipe 1) and return plates to room temperature.

12. Incubate the cells for an additional 3 days to allow for turnover of the targeted protein.

Note: If you suspect that your protein may have an exceptionally long half-life, incubating your cells with the dsRNA up to 6 days will allow sufficient time for more complete turnover of your protein.

Cell Extract Preparation

Note: Keep everything on ice from this point forward.

1. Harvest the cells by scraping with a cell lifter to dislodge cells from the dish and transfer them directly into 1.5-ml microcentrifuge tubes.

2. Pellet the cells by centrifugation at 1000g for 5 min and discard the supernatant.

3. Lyse the cells by adding 400 µl RIPA Lysis Buffer (Recipe 6), pipetting up and down to mix well.

4. Push the lysed cells through a 25-gauge, 1.5-inch needle 3 times. This ensures complete lysis of the cells.

5. Centrifuge at 107,000g for 30 min at 4°C to pellet cell debris.

6. Save the supernatant containing the whole cell extract and transfer to a new tube.

7. Store the cell extracts at -20°C.

Note: We typically analyze 5 µl to 10 µl of whole cell extracts for Western analysis. If you plan to immunoprecipitate your protein of interest, you may want to save an aliquot of the whole cell extract in an equal volume of LLB, 5× (Recipe 8) as a total lysate control. Cell extracts should be used immediately for immunoprecipitations, because some proteins cannot be immunoprecipitated after freezing.

Analysis of Cell Extracts

There are two different methods for determining the loss of your protein. Western analysis directly measures the loss of protein, but you must have an antibody directed against your protein (Fig. 2). If you do not have an antibody to your protein, you may indirectly measure the loss of protein by measuring the loss of mRNA. RNA will have to be isolated from the cells, and the probe for the Northern analysis must not contain any sequence that overlaps the dsRNA sequence.

Fig. 2.

Western analysis. Ten µl of RIPA cell extract were loaded per lane. WT lanes represent extracts from untreated S2 cells. RNAi lanes represent extracts prepared from S2 cells treated with dsRNAs directed against the indicated proteins (Dock, Ack, Lasp, and SH3PX1).

Note: Protocols for both Western and Northern analyses and total RNA isolation from cells can be found in most standard molecular biology laboratory manuals. Many aspects of these protocols must be empirically determined.

Notes and Remarks

We maintain a Web site for the Jack E. Dixon laboratory: (http://dixonlab.biochem.med.umich.edu/protocols/RNAiExperiments.html).

At this site we have an abbreviated protocol for the RNAi experiments and a FAQ page where commonly asked questions about the technique are listed and answered. We also share interesting unpublished observations by fellow users of the technique, and we would appreciate suggestions and comments.

References

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
View Abstract

Navigate This Article