Experiment Summary

In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells. The lentiviral expression plasmid is first engineered to contain the gene of interest driven by a TRE (tetracycline response element) promoter in a simple gateway cloning step and is then co-transfected into HEK293T cells, along with packaging and envelope plasmids to generate the virus. The virus is used to infect a cell type of interest at a low MOI so that the majority of the transduced cells contain a single viral integration. Infected cells are grown under selection, and viral integration is validated by qPCR. Gene expression in stably transduced cells is induced with doxycycline and validated by qPCR, immunoblot, and flow cytometry. This flexible lentiviral expression platform may be used for stable and robust induction of a gene of interest in a range of cells for multiple applications.

Materials and Reagents

A. Consumables and reagents

1. 10× PBS, store at 4°C
2. 293T culture dishes; 100 × 15 mm TC-treated Petri dishes
3. Centrifugal Filter Units
4. DMEM, store at 4°C
5. FBS, store at -20°C
6. G418, Geneticin, store at 4°C
7. Glutamine, store at 4°C
8. HEPES, store at 4°C
9. Lipofectamine 2000, store at 4°C
10. Opti-MEM, store at 4°C
11. Penicillin G, store at 4°C
12. Polybrene
13. Poly-L-Lysine Hydrobromide, store at 4°C
14. RPMI, store at 4°C
15. Sodium Pyruvate, store at 4°C
16. Petri dishes 100 mm × 15 mm

B. Plasmids

1. Entry Vector pEN_TmiRc3
2. Lentiviral expression vector pSLIK_Neo
3. pMDL
4. pRSV
5. pVSV

C. Cell lines

1. HEK293T cells
2. THP-1 cells

D. Primers

Equipment

1. Centrifuge
2. DNA Spectrophotometer
3. Flow Cytometer
4. Gel Imaging System
5. Protein gel tank
6. Real-time PCR system
7. Thermal Cycler
8. Transfer apparatus

Procedure

A. Prepare plates with Poly-L-Lysine

1. Dilute 20 mg/ml Poly-L-lysine (PLL) to 20 μg/ml PLL in 1× PBS by combining 50 μl of PLL (20 mg/ml) with 5 ml of 10× PBS and 45 ml of ddH₂O.
2. Add 4 ml of diluted PLL (20 μg/ml) to a 10 cm 293T culture dish.
3. Incubate for 1 h at 37°C.
4. Aspirate PLL solution and wash three times with 5 ml of 1× PBS. To store dishes, rinse three times with sterile ddH₂O and air-dry plates in a sterile tissue culture hood at room temperature.

B. Passage HEK293T cells

1. Aspirate media from a T75 flask of HEK293T cells at 90% confluency. One T75 flask should contain enough cells to plate three 10 cm dishes at 4.5 × 10⁶ cells per dish.
2. Wash flask with 5 ml of 2 mM EDTA PBS.
3. Add 5 ml of warm Trypsin-EDTA and gently rock the flask to detach cells.
4. Collect cells in a 15 ml conical tube and add 5 ml of complete media to inhibit trypsin.
5. Spin at 300 × g for 5 min.
6. Aspirate media and flick the pellet to loosen cells, resuspending in 10 ml of media and count cells.
7. Plate 4.5 × 10⁶ cells per PLL-coated 10 cm dish; cells should quickly adhere to the dish.

C. Transfect Cells

1. Change media on cells with 10 ml of regular growth media per plate 1 h prior to transfection.

2. Warm Opti-MEM (OM) at 37°C.

3. Dilute DNA constructs in OM to a total of 1.5 ml.

4. Dilute 60 μl of Lipofectamine 2000 (LF2000) with 1.44 ml OM. Let stand at room temperature for 5 min.

5. Add the 1.5 ml solution of LF2000/OM to the diluted DNA and let stand for 20 min at room temperature.

6. Carefully add 3 ml of DNA/LF2000/OM to the 10 ml media in the plate, one drop at a time.

7. Incubate overnight (12-18 h) at 37°C.

8. Aspirate media and replace with 10 ml of warm culture media per plate.

9. Incubate for an additional 36 h.

D. Virus Collection

1. Transfer media from 10 cm 293T culture plate to a 15 ml conical tube.
2. Centrifuge supernatant for 5 min at 500 × g at 4°C.
3. Filter supernatant through a 0.45 μm non-pyrogenic filter.
4. Store supernatant at 4°C while the Amicon unit is prepared.
5. Sterilize the Amicon unit by adding 15 ml of 70% ethanol to the filter cup. Let it sit for 10 min.
6. Centrifuge the Amicon unit for 15 min at 2,590 × g.
7. Discard ethanol from the collection tube.
8. Add 15 ml of sterile water to the filter cup. Let it sit for 5 min.
9. Centrifuge the Amicon unit for 15 min at 2,590 × g. Discard water from the collection tube.
10. Add the viral supernatant to the Amicon unit.
11. Centrifuge for 30 min at 2,590 × g at 4°C. The supernatant should completely pass through the filter, with none remaining in the filter cup portion.
12. Add 1 ml of RPMI to the filter cup. Collect the viscous concentrated virus and store at 4°C.

E. Lentivirus titration in 293T cells

1. Plate 293T cells harvested from log phase cultures in 12-well tissue culture plates at a concentration of 1.44 × 10⁵ cells/well and incubate overnight. Visually confirm that the 293T cells are healthy, evenly distributed, and at 40-50% confluence at the time of infection.
2. Aspirate medium from 293T cells and add 0.9 ml of pre-warmed 293T growth medium containing 8 µg/ml polybrene. Return plates to incubator.
3. Dilute viral solutions in 293T growth medium containing 8 µg/ml polybrene. A final volume of 100 µl is needed for each dilution, and each sample will be diluted another 10-fold when added to the cells. A set of serial dilutions (usually three) over the range 10-10³ is tested for unconcentrated virus, and a range of 10²-10⁵ is tested for concentrated virus.
4. Add 100 µl of each virus dilution to wells of 293T cells (in 0.9 ml of media). To serve as matched uninfected controls, add 100 µl of 293T growth media/polybrene that does not contain virus to several wells. Incubate cells for 24 h.
5. After 24 h, replace the media with fresh 293T growth medium and return plates to the incubator.
6. Harvest cells for flow cytometric analysis 48-72 h after infection. Pool the media supernatant, washes, and dislodged cells from each sample well into FACS tubes to ensure that all cells are included.
7. Pellet cells by centrifugation (300 × g, 5 min, 4°C).
8. Determine the percentage of Venus-positive cells by flow cytometric analysis by comparison of infected samples with uninfected 293T cells. Identify samples in which the infection rate is 3-10% and calculate the number of cells infected on the day of infection. This is approximately equivalent to the number of infectious virus particles added per well, assuming a 1:1 infection ratio. The number of active viruses detected is used with the volume and dilution of virus stock to calculate the virus concentration in the stock solution. This provides the lentiviral titer with respect to 293T cell infection.

Lentiviral titer = number of infected cells × volume of virus stock × dilution of virus stock

F. THP-1 Cell Transduction

1. Calculate viral particles per milliliter by dividing 1.175 × 10⁷ by the total volume of concentrated virus recovered per plate.

2. Calculate the volume of virus needed to infect cells at 10 MOI by dividing 5.0 × 10⁶ by the concentration of viral particles per ml. We typically infect 0.5 × 10⁶ THP-1 cells with an approximate MOI of 10 (293T transduction units).

Volume of virus (ml) = (number of cells per well × MOI)/viral particles per ml, i.e.(0.5 × 10⁶ × 10)/viral particles per ml.

This equates to an MOI of<1 for THP-1 cells, usually around 30% transduction efficiency [where transduction efficiency (%) = number of transduced cells/total number of cells]. Transduction efficiency can be measured by FACS to count the number of THP-1 cells that are positive for expression of the lentivirus-encoded transgene or a constitutively expressed Venus transduction marker as a fraction of the total number of cells. We aim for this relatively low infectivity to ensure that most cells do not have multiple viral integrations.

3. Plate 0.5 × 10⁶ cells per well into two wells of a 24-well dish.

4. Infect cells 0.5-3 h after seeding.

5. Infect one well by diluting virus up to 500 μl with serum-free growth medium supplemented with 4 μg/ml polybrene and 100 U/ml penicillin G.

6. Aspirate media and replace it with diluted virus.

7. After 4 h, add 1.5 ml regular growth media with penicillin.

8. The next day, split cells and add 1 × 10⁶ cells into Valmark dishes.

9. One to two days after splitting, select for cells by growing in complete media supplemented with G418 (1 mg/ml).

G. Gene Expression Validation in THP-1 Cells

1. Plate cells in a 6-well plate and incubate at 37°C for 6 h.
2. Add DOX to the cells (1 μg/ml) and incubate for 6-18 h.
3. Harvest cells and isolate RNA or protein.
4. Validate viral integration by qPCR (Figure 1B) and DOX-induced overexpression by qPCR, immunoblot, or flow cytometry.

Figure 1. Lentivirus-mediated conditional expression of NOD1 and NLRP2.