Combined Adenoviral Transduction and siRNA-Mediated Knockdown in Cancer Cells

Abstract

Source: Fuchs, M. et al. Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments. J. Vis. Exp. (2016)

This video demonstrates adenovirus-mediated delivery of a fluorescent transgene combined with siRNA–calcium phosphate transfection in cervical cancer cells. The adenovirus expresses a fluorescent replacement protein, while the siRNA induces targeted mRNA degradation through RISC binding. This sequential co-transfection enables simultaneous knockdown of the endogenous protein and functional rescue by the transgene, allowing visualization and confirmation of protein replacement through fluorescence imaging.

Protocol

1. Adenovirus Transduction and Endogenous Protein Knockdown by Small Interfering RNA (siRNA) Transfection in HeLa-RFP-H2B Cells Using Calcium Phosphate (CaPi) Precipitates

NOTE: A control adenovirus carrying an unrelated gene (i.e., LacZ) or no gene is used to reach a minimal multiplicity of infection (MOI) in all Adenofections (10-20 plaque-forming units (PFU)/cell) using the lowest amount of recombinant adenovirus carrying the gene of interest.

NOTE: This procedure has been shown to help normalize expression per cell in a large cell population.

1. One day after cell plating, count the number of cells from the additional plated dish. Aspirate the medium and rinse once with 1 mL 0.05% Trypsin/ethylenediaminetetraacetic acid (EDTA). Add again 1 mL of 0.05% Trypsin/EDTA and incubate at 37 °C, 5% CO₂ until all cells have detached. Separate the cells well using a 1 mL pipette and count the number of cells per mL using a haemocytometer.
2. Determine the quantity of virus needed to transduce cells at a MOI of 2 PFU of the protein of interest (POI) per cell and 18 PFU per cell of an empty vector (e.g., LacZ) to have a total of 20 PFU per cell. At the same time, transduce 4 PFU of each baculovirus encoding green fluorescent protein (GFP)-tagged actin and red fluorescent protein (RFP)-tagged α-tubulin. Transduce the cells using the following adenofection protocol.
3. Prepare in a sterile hood a 1.5 mL plastic tube for each condition and add 400 μL of warm alpha minimum essential medium (αMEM).
4. Thaw an aliquot of the viruses slowly on ice and, if necessary, dilute the virus stock in order to pipet a volume greater than 1 μL to minimize pipetting errors.
5. Add the calculated virus quantity per condition to each 1.5 mL plastic tube containing 400 μL αMEM and mix gently by pipetting. Place the virus stock immediately back at -80 °C to keep its activity.
6. Aspirate the medium from the cells and gently pipette the virus mix dropwise. Incubate the cells at 37 °C, 5% CO₂ and agitate the plates carefully under the sterile hood every 15 min for 1 hr to ensure that the cells are well covered with the virus. Using a small amount of medium facilitates the contact of the virus with the cells.
7. After incubation, gently pipette 1.6 mL αMEM to each plate to obtain a total volume of 2 mL, start cell synchronization by adding 2 mM thymidine and incubate the cells for a further 2 hr at 37 °C, 5% CO₂.
8. Meanwhile, prepare the siRNA transfection mix following the CaPi transfection method (see Figure 1). If no siRNA is necessary, replace the amount of siRNA by sterile water to perform an empty transfection.
9. Calculate 200 μL mix for each condition, resulting in 400 μL per duplicate. The following example is given for a final siRNA concentration of 50 nM and has to be adapted for each protein of interest. In a 1.5 mL plastic tube, pipette 50 μL 1 M CaCl₂ and 11 μL 20 μM siRNA into 139 μL sterile H₂O and mix by vortexing. After a quick spin, carefully add 200 μL 2× HEPES-buffered saline (HBS2x) (280 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.5 mM Na₂HPO₄, pH 7.01-7.05) dropwise.
   NOTE: The smaller the drops, the smaller the precipitates, resulting in better transfection efficiency. Gently mix three times by air injection using a 200 μL pipette.
10. Incubate the mix for 30 min at room temperature (RT). Slowly add 200 μL of the transfection mix to each plate and agitate cross-wise. Transfer the plates to 37 °C, 5% CO₂ for 16 hr.
11. The next day, rinse cells twice with 2 mL warm HEPES (6.7 mM KCl, 150 mM NaCl, 10 mM HEPES, pH 7.3) and add 2 mL αMEM. Do not proceed with more than four cell plates at a time since variations in temperature affect the length of the cell cycle.
12. Visualize the infection efficiency under an inverted fluorescent microscope at a magnification of 20-40x (air) and acquire three representative images per condition in both fluorescent and transmission channels for documentation. Seven hours later, add 2 mM thymidine and incubate for a further 16 hr at 37 °C, 5% CO₂.
13. The following day, 48 hr after siRNA transfection and virus infection, rinse the cells twice with 2 mL warm phosphate-buffered saline (PBS) and release for 7 hr in 2 mL αMEM without Phenol Red for live cell imaging or with Phenol Red for protein extraction.
14. Harvest cells for each condition 48 hr post-transfection for preparation of protein extracts and Western blot analysis to determine the efficiency of knockdown and the expression of the endogenous protein.
    NOTE: Use antibodies against the protein of interest as well as appropriate antibodies serving as loading controls. The efficiency of knockdown should be determined by loading decreasing amounts of control cell lysates (transfected with control siRNA) to provide a titration curve (i.e., 1, ½, ¼, ⅛).

Results

Figure 1. Planning of a typical adenofection protocol. Successive steps of a typical experiment are shown for the protocol using CaPi precipitates (underlined in grey) or the protocol using a cationic lipid transfection reagent (highlighted in yellow), including cell plating, transduction of adeno- and baculoviruses, transfection of siRNA duplexes, and cell synchronization with a double thymidine block. Time lines in hours for both protocols are shown on the right side of the figure.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EDTA	Sigma	E5134
Fetal Bovine Serum (FBS)	Thermo Fisher	12483-020
Fibronectin	Sigma	F1141
Glass bottom dishes, 35 mm	MatTek Corperation	P35G-1.5-20-C Case
HeLa-RFP-H2B	Kind gift of Dr Sabine Elowe, Québec, Canada
HEPES	Fisher Scientific	BP310-1
KCl	Fisher Scientific	BP366-500
L-Glutamine	Thermo Fisher	25030081
NaCl	Fisher Scientific	BP358-10
Minimal Essential Medium (MEM) Alpha	Wisent	310-101-CL
Minimal Essential Medium (MEM) Alpha without Desoxyribonuleosides/Ribonucleosides	Thermo Fisher	12000-022
Minimal Essential Medium (MEM) Alpha without Phenol Red	Thermo Fisher	41061-029
Na₂HPO₄	Biobasic	S0404
Adenovirus custom design	Welgen	Custom design
Calcium Chloride	Fisher Scientific	C79-500
CellLight® Actin-GFP, BacMam 2.0	Thermo Fisher	C10582
CellLight® Tubulin-RFP, BacMam 2.0	Thermo Fisher	C10614
siRNA duplexes	Dharmacon	Custom design
Thymidine	Sigma	T9250
Trypsine 2.5%	Thermo Fisher	15090-046