For more product literature on the Open Biosystems human ...



For more product literature on the Open Biosystems human and mouse shRNAmir libraries, please visit the Open Biosystems website at:



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We use the Phoenix packaging cell line to produce infectious retroviral supernatant.

The Phoenix cell line produces amphotrophic retrovirus that infects a broad range of mammalian cells.  This cell line is derived from the human embryonic kidney (HEK 293) cell line. (The viral gag, pol, and VSV-G genes necessary for particle formation and replication are stably integrated into the genome using separate selection markers.  Therefore, expression of packaging proteins can be fairly well maintained by periodically culturing the cells in media containing the corresponding antibiotics.)  

It is a helper free line which produces infectious but replication incompetent virus.

We do not measure the viral titer, however, viral titers are in the range of 10^5 or 10^6 IU/ml.

Graphic of retroviral particle production:

[pic]

Getting Started with shRNA

Expression Arrest shRNA Bacterial Glycerol Stocks offer a renewable resource for shRNA constructs. Individual clones may be propagated and prepped for plasmid DNA. The vectors can be used directly for transient or stable transfection or used with packaging plasmids in a packaging cell line to produce retroviral transduction particles.

Culture conditions for individual plasmid preparations

Most plasmid mini-prep kits recommend a culture volume of 1–10 ml for good yield.

For shRNA constructs, 5ml of culture can be used for each mini-prep; generally producing from 5–20 ug of plasmid DNA.

1. Using a sterile loop or a pipette tip, streak the shRNA culture onto a LB agar plate containing 50 ug /ml Chloramphenicol. Incubate the plate overnight at 37°C.

2. The following day, pick 3 to 5 well-isolated colonies from the agar plate and inoculate 6 ml of the 2XLB Chlor50+Kan25. Incubate at 37°C for 14-16 hrs with vigorous shaking (300rpm).

5. The following day remove 1 ml of the culture and place in a sterile 2-ml sterile microcentrifuge tube. Place this tube at 4°C until the plasmid DNA from the remaining culture has been analyzed.

6. Pellet the remaining 5-ml culture and prepare of plasmid DNA according to kit instructions.

7. Run 3-5ul of the plasmid DNA on a 1% agarose gel. The uncut pSM2 shRNA constructs run at about 7-10kb while the most common product of a

recombination event will run at ~1.5-1.8kb. Pick the plasmid DNA from a colony in which recombination is not present in a significant amount for transfection.

8. Prepare an 8% glycerol stock culture using the 1ml of culture you removed prior to plasmid preparation.

We recommend preparing ultra-pure DNA to ensure both high purity and low endotoxin levels as required for transfection into eukaryotic cells. We then recommend using lipofectamine 2000 or an equivalent transfection reagent to transiently or stably transfect the plasmid DNA into either target cells or a packaging cell line (preferred method) to produce retroviral transduction particles.

Transfect packaging cells:

One day prior to transfection:

Seed packaging cells in a tissue culture plate (60mm in diameter) at a density which will yield a cell density about 80% of confluency the next day. Incubate overnight in complete growth medium.

1.) Day 1:

Set up transfection as per manufacturer’s suggestions.

Note: I use lipofectamine 2000 tranfection reagent at a 1:2.5 DNA:lipo ratio. I use 5ug of DNA for a 6 cm plate and 12.5ul lipofectamine. I follow maunfacturer’s instructions exactly as for set up, incubations, etc. I do vary a little bit in how I add the transfection reagent to cells. I aspirate all media from cells and add only the 500ul DNA:lipofecamine complex. I incubate ~2-3 min (at either room temp. or back in the 37°c incubator) and then add 3.0ml complete cell media and return cells to the incubator overnight.

Incubate cells overnight at 37°c.

2.) Day 2:

Remove medium and add 3ml fresh DMEM media supplemented with 10% FCS. Incubate an additional 24-48 hrs at 37°c (or at 32° for greater virus stability.)

Note: I have found that there is very little difference in viral titer at 48hrs vs. 72hrs. However, if I’m not collecting virus for 72hrs I will definitely incubate cells at 32°c for the last 24 hrs. The half-life is much longer at 32°c!

3.) Day 4-5:

Harvest virus-containing supernatants and remove any cells from harvested medium by filtration through 0.45 µm cellulose acetate (low protein binding) syringe filter. This is the viral stock. At this point the virus can be used directly to infect target cells or stored at –70°C (one cycle of freeze-thaw will reduce the titer 2-3 fold).

Infection of Target Cells

! Note: We and many others have found that "spinning" the virus onto cells can result in up to a ten-fold increase in effective titer of the virus. The following is the protocol for "Spin-fection" and is highly recommended.

Infect cells:

1.) On the day of infection, remove existing media from the target cells which should be at ~70% confluence in a 6 or 12 well dish.

2.) Apply the virus supernatant + 4ug/ml polybrene* directly on top of cells. The volume should completely cover the cells (ie. 1.0ml for 6 well plate.)

3.) Seal plate and place in a microtiter rotor and spin at 1800 rpm for 45 minutes at room temperature.

Note: Spinning cells at 1800 rpm for 45 minutes is not enough to sediment free virus. It is thought that virus on membrane fragments is spun onto cells in a manner which effects greater infection.

4.) Return plate to incubator and incubate for 3-6 hours at 37°c / 5% CO2.

5.) Optional: Repeat spin infection at 1800rpm for 45 min. at room temperature.

6.) Replace media with normal target cell media. Incubate overnight at 37°c.

7.) The next day, split the cells at least 1:5, let attach for 1 day.

8.) Replace media with puromycin selection media (50 -4000ng/ml** puromycin in normal target cell complete medium.)

9.) Replace media with puromycin selection media every 3-4 days. Select for resistant colonies which should be visible in 7-12 days.

* Polybrene promotes infection by preventing electrostatic repulsions that can occur between the virus and the cell membrane. The use of polybrene is recommended in every protocol I have seen. However, in our lab, side by side experiments with and without polybrene have shown no real difference in infection rates.

** cell type, infection with retrovirus, transfection with plasmid, cell health, culture medium, and growth conditions can all effect the optimal puromycin concentration for selection. It is highly recommended that you first perform a puromycin titration (kill curve.)

PROTOCOL Puromycin Kill Curve and Puromycin Selection

In order to generate stable cell lines, it is important to determine the minimum amount of puromycin required to kill non-transfected/transduced cells. This can be done by generating a puromycin kill curve.

Puromycin Kill Curve procedure:

1. On day 0 plate 5 x 104 cells per well in a 24 well plate in enough wells to carry out your puromycin dilutions. Incubate overnight.

2. Prepare media specifically for your cells containing a range of antibiotic, for example: 0 - 15μg/ml puromycin.

3. The next day (day 1) replace the growth media with the media containing the dilutions of the antibiotic into the appropriate wells.

4. Incubate at 37°C.

5. Approximately every 2-3 days replace with freshly prepared selective media.

6. Monitor the cells daily and observe the percentage of surviving cells. Optimum effectiveness should be reached in 3-6 days under puromycin selection.

7. The minimum antibiotic concentration to use is the lowest concentration that kills 100% of the cells in 3-6 days from the start of antibiotic selection.

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