Getting Started with shRNA in the pGIPZ lentiviral vector



Getting Started with shRNA in the pGIPZ Lentiviral Vector

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 HEK 293T to produce lentiviral transduction particles.

PRODUCT DESCRIPTION

The GIPZ lentiviral shRNAmir library was developed by Open Biosystems in collaboration with Dr. Greg Hannon (CSHL) and Dr. Steve Elledge (Harvard). This library combines the design advantages of microRNA-adapted shRNA (shRNAmir) with the pGIPZ lentiviral vector to create a powerful RNAi trigger capable of producing RNAi in most cell types including primary and non-dividing cells.

VECTOR INFORMATION

Versatile vector design Features of the pGIPZ™ lentiviral vector that make it a versatile tool for RNAi studies include:

• Ability to perform transfections or transductions using the replication incompetent lentivirus (Shimada, et al. 1995)

• TurboGFP and shRNAmir are part of a bicistronic transcript allowing the visual marking of shRNAmir expressing cells

• Amenable to in vitro and in vivo applications

• Puromycin drug resistance marker for selecting stable cell lines

• Molecular barcodes enable multiplexed screening in pools

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



PROTOCOL - PLASMID PREPARATION

Culture conditions for individual plasmid preparations For plasmid preparation, grow all pGIPZ™ clones at 37°C in 2X-LB broth (low salt) media plus 100μg/ml carbenicillin ( or ampicillin) only.

2X-LB broth (low salt) media preparation

LB-Broth-Lennox 20g/l

Peptone 10g/l

Yeast Extract 5g/l

Appropriate antibiotic(s) at recommended concentration(s)

Most plasmid mini-prep kits recommend a culture volume of 1-10ml for good yield. For shRNAmir constructs, 5ml of culture can be used for one plasmid mini-prep generally producing 10-15µg of plasmid DNA.

1. Upon receiving your glycerol stock(s) containing the shRNAmir of interest store at -80°C until ready to begin.

2. To prepare plasmid DNA first thaw your glycerol stock culture and pulse vortex to resuspend any E. coli that may have settled to the bottom of the tube.

3. Take a 10μl inoculum from the glycerol stock into 5ml of 2X-LB (low salt) with 100μg/ml carbenicillin. Return the glycerol stock(s) to -80°C.

Note: If a larger culture volume is desired, incubate the 5ml culture for 8 hours at 37ْC with shaking and use as a starter inoculum. Dilute the starter culture 1:500-1:1000 into the larger volume.

4. Incubate at 37°C for 18-19 hours with vigorous shaking.

5. Pellet the 5ml culture and begin preparation of plasmid DNA following kit instructions.

6. Run 5μl of the plasmid DNA on a 1% agarose gel to check for DNA quality and recombination. pGIPZ with shRNAmir is 11774bp.

Note: Due to the tendency of all viral vectors to recombine, we recommend keeping the incubation times as short as possible and avoid subculturing. Return to your original glycerol stock for each plasmid preparation.

shRNA Lentivirus Production using HEK293T cells

note: Biohazard Concerns

Lentivirus is a modified HIV virus and although unable to replicate in a host, it must be handled with caution. When working with these viruses, work only in BL2+ designated hoods or viral vector rooms. All handling, storage and disposal of biohazard waste must be in accordance with Institute rules and regulations.

Materials

1.) HEK293T cells

2.) Plasmid DNA for:

a.) shRNA-pGIPZ plasmid

b.) psPAX2 (or pCMV-dR8.74) (plasmid containing gag, pol and rev genes)

c.) pMD2.G (VSV-G expressing envelope plasmid)

d.) pGIPZ non silencing control plasmid

3.) FuGENE 6 or Lipofectamine 2000

Procedure

Note: I use lipofectamine 2000 at a 1:2.5 DNA to lipofectamine ratio. The following protocol has been optimized for lipofectamine2000 transfection in a 6 cm dish. The procedure may differ slightly with different transfection reagents.

1 day before transfection:

1.) Split 293T cells into culture dishes at ~80% confluence. Be sure cells are evenly distributed throughout the plate. Clumps will reduce transfection efficiency.

2.) Incubate for next 24 hours undisturbed at 37ºC and 5% C02.

Day of transfection:

Follow transfection protocol for the reagent you are using or use the following protocol for lipofectamine 2000 reagent.

1.) Add 2.5ug plasmid DNA from your of your pGIPZ shRNA, 2.5ug psPAX2 (or pCMV-dR8.74), 1.0ug pMD2.G to 250ul DMEM (no serum or antibiotics)

2.) Add 15ul lipofectamine (1 : 2.5 DNA to lipofectamine ratio) to 250ul DMEM (no serum or antibiotics)

3.) Incubate for 5 min. at RT

4.) Combine the tubes from steps 1 and 2 and mix gently by flicking to mix

the contents. Incubate the combined mixture at room temperature for 20 minutes.

5.) Aspirate media from 293T cells and carefully add the 500ul DNA / Lipo complex to the dish.

Note: 293T cells detach very easily from the plate. Take care in adding the complex mix.

6.) Swirl the plate very gently to mix. Incubate a few minutes (~2-3 min.) at room temperature and then very carefully add 3mls normal 293T media (DMEM media supplemented with 10%FBS and 1% Pen/Strep) to cells.

7.) Incubate the cells overnight at 37ºC and 5% C02.

Day 1 (24 hours) after transfection:

1.) Change the media to fresh DMEM media supplemented with

10% FBS and 1% Pen/Strep. Incubate an additional 24 hrs at 37ºC / 5% CO2 (or at 32° for greater virus stability.)

Note: 293T cells can detach easily from the plate and you do not want to disturb them, particularly at this point. Add the fresh media carefully.

Day 2 (48 hours) after transfection:

Note: Examine the cells microscopically for the TurboGFP expression. If they are expressing GFP, you can assume the cells have taken in the DNA and are producing virus.

1.) Collect virus-containing supernatant and remove any cells from harvested medium by filtration through 0.45 µm cellulose acetate (low protein binding) syringe filter.

Store virus overnight at 4°c

2.) Add another 3.0 – 3.5ml of fresh DMEM media supplemented with 10% FBS and 1% Pen/Strep to the cells. Incubate the cells overnight at 37ºC / 5% CO2 (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!

Day 3 (72 hours) after transfection:

1.) Collect virus-containing supernatant and remove any cells from harvested medium by filtration through 0.45 µm cellulose acetate (low protein binding) syringe filter. Combine with 48hr supernatant. This is the viral stock.

Note: Virus can be used directly to infect target cells, or stored at 4°c for use within a week. For longer term storage virus can be stored at -70°c. Titer will decrease 2-3 fold with each freeze / thaw so immediate use is recommended.

2.) Discard the 293T cells.

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%-80% 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. 1.0 ml 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.) Add at least 1 ml normal target cell media for a total volume of >2 ml. Incubate overnight at 37°c.

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

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

8.) Replace media with puromycin selection media every 3-4 days. Select for resistant colonies.

* 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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