[민유홍] RevTet-Off System Protocol

RevTet-Off System Protocol

2007. 6.20 Yu-Hong Min

Guidelines for working with retroviruses

Put on double gloves.

All waste must be autoclaved.

Liquid waste : Do not suction. Collect in glass bottle then autoclave.

PT67 packaging cell culture

DMEM, 10% tetracycline-free FBS, 1% P/G

Doxycycline

Dissolve at 1 mg/ml in water and filter sterilize. Store at 4°C in the dark, and use within four weeks. Aliquots can be frozen at –20°C for long-term storage. This stock can be frozen/thawed up to five times. Fresh Dox should be added to the media each time, rather than keeping media with added Dox in the refrigerator.

I. Titrating G418 and hygromycin concentrations

1. Plate 1 X 10⁵ target cells (e.g., A549) in each well of a 6 well plate.

2. Incubate with complete medium plus 0, 50, 100, 200, 400, 800 μg/ml of G418 or hygromycin for 7-10 days, replacing selective medium every 4 days.

3. Select lowest concentration that kills all the cells within one week.

Optimal concentrations

PT67 : G418 0.8 mg/ml, hygromycin 0.4mg/ml

A549 : G418 0.5 mg/ml, hygromycin 0.4mg/ml

II. Selection of stable virus-producing cell lines

1.     Plate 8 X 10⁵ PT67 cells in a 10 cm culture dish

2.     Next day, transfect cells with 24 μg of DNA (pRevTet-Off or pRevTRE-Luc or pRevTRE-p43 or pRevTRE-p38 or pRevTRE-DX2 or pRevTRE-p18) using 48 μl Lipofectamine 2000. After 3h, change the medium.

3.     2 days after transfection, replace the medium with selection medium (complete medium with G418 0.8mg/ml for pRevTet-Off, or hygromycin 0.4mg/ml for pRevTRE).

4.     After sufficient number of resistant cells were grown, prepare frozen stocks of the selected cell populations.

III. Establishing a stable Tet-Off cell line

A. Collection of medium from the packaging cells

1.     Plate each PT67 cells stably transfected with pRevTet-Off or pRevTRE-Luc in 10 cm culture dish.

2.     When the confluency is ~ 70%, replace the medium with new complete medium.

3.     After 24h, collect the medium, filter through a 0.45 μm filter. Use a nitrocellulose acetate or polysulfonic (low protein binding) filter but not nitrocellulose. Use for infection immediately, or store 4℃ for several days, or -70℃ for up to one month (viral titer may fall 2-4 fold after freezing).

B. Infection and selection

1.     Plate target cells (e.g., A549) one day before infection in a 10cm dish to attain a confluency of 40 ~ 60% at the time of infection.

2.     Combine 0.1 ~ 1ml of filtered medium from PT67-pRevTet-Off, and ~10 ml of new complete medium for target cells. Add polybrene (Hexadimethrine bromide) to a final concentration of 4 μg/ml. Incubate with target cells for 24 h.

3.     2 to 3 days after the infection of target cells, subject cells to G418 treatment (concentration determined previously) for more than 7 days. Change medium as necessary.

4.     Once clones are visible, isolate large, healthy colonies and transfer them to individual wells of 12 well or 24 well plate by using cloning cylinders or just pipett tip.

C. Screening for inducible clones with RevTRE-Luc virus

1.     Expand each clone as needed to perform infections.

2.     For each G418-resistant clone, maintain a stock plate, and set up 2 additional cultures in wells of a 6 well plate to use as a test plate for screening. Plate test cells one day before infection. Plate a sufficient number of cells to attain a confluency of 40-60% at the time of infection.

3.     Combine 1 ~ 3ml of 0.45 μm-filtered medium from PT67-pRevTRE-Luc, and ~10 ml of new complete medium for target cells. Add polybrene to a final concentration of 4 μg/ml. Incubate for 24 h.

4.     Change medium and add doxycycline (final 1-2 μg/ml) to one of the two test plates.

5.     Incubate the cells for 48h.

6.     Assay for luciferase and calculate fold-induction (= -Dox RLU/+ Dox RLU):

7.     Select clones with the highest fold-induction (highest expression with lowest background).

8.     Expand each clones and freeze stocks.

IV. Establishing a double-stable, inducible cell line

A. Collection of medium from the packaging cells

1.     Plate each PT67 cells stably transfected with pRevTRE-p43, p38, DX2 or p18 in 10 cm culture dish.

2.     When the confluency is ~ 70%, replace the medium with new complete medium.

3.     After 24h, collect the medium, filter through a 0.45 μm filter. Use a nitrocellulose acetate or polysulfonic (low protein binding) filter but not nitrocellulose. Use for infection immediately, or store 4℃ for several days, or -70℃ for up to one month (viral titer may fall 2-4 fold after freezing).

B. Infection and selection

1.     Plate selected G418-resistant and doxycycline-inducible clone one day before infection in a 10cm dish to attain a confluency of 40 ~ 60% at the time of infection.

2.     Combine 0.1 ~ 1ml of 0.45 μm-filtered medium from PT67-pRevTRE-p43, -p38, -p18 or -DX2), and ~10 ml of new complete medium for target cells. Add polybrene to a final concentration of 4 μg/ml. Incubate with target cells for 24 h.

3.     2 to 3 days after the infection of target cells, subject cells to G418, hygromycin (concentration determined previously) and doxycycline (final 1 μg/ml) treatment for more than 7 days. Change medium every 2 days.

4.     Once clones are visible, isolate large, healthy colonies and transfer them to individual wells of 12 well or 24 well plate by using cloning cylinders or just pipett tip.

5.     Expand each clone with G418, hygromycin and doxycycline.

C. Screening for inducible clones

1.     For each G418 and hygromycin-resistant clone, maintain a stock plate, and set up 2 additional cultures in wells of a 6 well plate to use as a test plate for screening. Plate test cells one day before infection. Plate a sufficient number of cells to attain a confluency of 40-60% at the next day.

2.     Change medium and add complete medium without doxycycline to one of the two test plates.

3.     Allow the cells to grow for at least 48h, then assay each sample for gene expression using Western blotting or RT-PCR.

4.     Select clones with the highest induction. Prepare frozen stocks.

V. Determination of effective concentrations of doxycycline

1.     For the double-stable and inducible clones, plate a sufficient number of cells in 6 well plates to attain a confluency of 40-60% at the next day.

2.     Add doxycycline to final concentrations of 0, 0.001, 0.01, 0.1, 1.0, 10, 100, 1000 ng/ml.

3.     Allow the cells to grow for 48h.

4.     Assay each sample for inhibition by using Western blotting or RT-PCR.