Lentiviral transduction

Materials and methods

Day 1:

1) HEK293 (ATCC, catalog number: CRL-1573)

1) DMEM + 10% FBS + 2% L-Glutamine + 1% Sodium Pyruvate (D10 media)

-       DMEM (Hyclone, catalog number: SH30081.01)

-       Sodium Pyruvate (ThermoFisher Scientific, catalog number: 11360-070)

-       FBS (ThermoFisher Scientific, catalog number: 26140079)

-       L-Glutamine (Lonza, catalog number: 17-605E)

2) Phosphate-buffered saline (1X)

3) 0.025% Trypsin-EDTA (Lonza, CC-5012)

4) Hemocytometer (Paul Marienfeld, catalog number: 0680030)

5) 15 cm cell culture dishes (Corning, catalog number: CLS430599)

6) 1.5 ml Eppendorf tubes (SSI, catalog number: SSIB1210-00S)

7) 10 ml pipette (Greiner Bio, catalog number: 606180)

8) 15 ml falcon tubes (Greiner Bio, catalog number: 188271)

Day 2:

1) DMEM + 2% L-Glutamine + 1% Sodium Pyruvate (Serum-free DMEM)

-       DMEM (Hyclone, catalog number: SH30081.01)

-       Sodium Pyruvate (ThermoFisher Scientific, catalog number: 11360-070)

-       L-Glutamine (Lonza, catalog number: 17-605E)

2) Phosphate-buffered saline (1X)

3) FBS (ThermoFisher Scientific, catalog number: 26140079)

4) Lipofectamine 2000 (ThermoFisher Scientific, catalog number: 11668030)

5) Helper plasmids

     -    psPAX2 (Addgene plasmid #12260)

     -    pCMV-VSV-G (Addgene, plasmid #8454)

6) Plasmids of interest

     -    FUGW-Hebp-1

     -    FUGW (Addgene, plasmid #14883)

7) DMEM-F12 media (ThermoFisher Scientific, catalog number: 11330-032)

8) 15 ml falcon tubes (Greiner Bio, catalog number: 188271)

Day 3:

1) 50 ml syringes (Therumo, catalog number: 191022T)

2) 0.45 µm syringe filters (Minisart, catalog number: 16537)

3) 50 ml falcon tubes (Greiner Bio, catalog number: 227261)

4) 100K Amicon Ultra-15 (Merck Millipore, catalog number: UFC910096)

5) 1.5 ml eppendorf tubes (SSI, catalog number: SSIB1210-00S)

Procedures

Day 1:

1) Pre-warm D10 media and trypsin-EDTA in a 37^oC water bath.

2) Check HEK293 cells in a 15 cm dish under a light microscope and ensure that they are at least 90% confluent before subsequent steps.

3) Aspirate media and wash cells gently with 2 ml of PBS.

4) Aspirate PBS and add 2 ml of trypsin-EDTA and incubate for 1 minute at 37^oC.

5) To neutralize trypsin-EDTA, add 4 ml of D10 media to the cells. General rule of thumb is to add 2X the volume of D10 media to volume of trypsin used.

6) Mix gently by aspirating up and down using a 10 ml stripette. The cells should dislodge from the bottom of the plate.

7) Collect the cells in a 15 ml falcon tube and spin cells at 1000 rpm for 4 minutes at room temperature (25^oC).

8) After spinning, aspirate out the media and resuspend cell pellet in 5 ml of D10 media. Gently mix by pipetting up and down.

9) Add 10 µl of cells to 190 µl of D10 media in a 1.5 ml Eppendorf tube and mix gently. Ideally, we recommend a 20X dilution of the cells.

10) Prepare hemocytometer by wiping with 70% ethanol (EtOH). Pipette 10 µl of the diluted cells onto one of the chambers of the hemocytometer and cover with a glass slide. Make sure that the cells spread out and that the glass side sticks to the hemocytometer.

11) Count 4 of the 4x4 quadrants and take the average cell count.

      For example:

Average cell count over 4 quadrants = 190 cells

Average = 190/4 = 47.5

Cell count = 47.5 x 10⁴ x dilution factor (in our case, 20)

                   = (47.5 x 10⁴) x 20 = 9.5 x 10⁶ cells/ml

12) Seed four 15 cm plates with 13 million (13 x 10⁶) cells per plate. We recommend 2 plates for the preparation of one virus. Based on our previous count, you will require the following volume of cells per plate:

Volume of cells = 13 x 10⁶ cells / 9.5 x 10⁶ cells/ml = 1.37 ml

13) To each fresh 15cm plate, add required amount of cells and top up with 12mL of D10 media.

14) Keep cells in a humidified incubator (37^oC, 5% CO₂).

Day 2:

1) Thaw helper plasmids and plasmids of interest at 4^oC. Spin briefly in a centrifuge if necessary.

2) Pre-warm serum-free DMEM and FBS in a 37^oC water bath.

3) Check seeded HEK293 cells under a light microscope and ensure that they are at least 70% confluent before subsequent steps.

4) For each plate to be transduced, prepare:

     -    8 ml of serum-free DMEM with 3% FBS

     -    2 ml of serum-free DMEM for DNA mix

     -    2 ml of serum-free DMEM for Lipofectamine 2000 solution

5) Prepare DNA mix with helper plasmids and plasmid of interests in 2 ml of serum-free DMEM in a 1:1:2 ratio.

For example:

To prepare FUGW-Hebp-1 DNA mix, dilute 27.0 µg of FUGW-Hebp-1 plasmid with        13.5 µg of psPAX2 and 13.5 µg of pCMV-VSV-G in 2 ml of serum-free DMEM.

6) Lipofectamine 2000 solution is prepared at a ratio of 1 µg DNA: 1.2 µl of Lipofectamine 2000. Since, each plate will get a total of 54 µg of plasmid, 64.8 µl of Lipofectamine 2000 should be added to 2 ml of serum-free DMEM for preparing Lipofectamine 2000 solution.

7) Combine the DNA mix and Lipofectamine 2000 solution (4 ml in total) and mix gently. Incubate further at room temperature (25^oC) for 20 minutes.

8) Aspirate out D10 media from seeded HEK293 cells in a 15 cm dish and add 8 ml of DMEM with 3% FBS.

9) Add 4ml of DNA/Lipofectamine 2000 solution mix dropwise to each 15 cm plate.

10) Keep cells in a humidified incubator (37^oC, 5% CO₂) for 6 to 8 hours.

11) Pre-warm DMEM-F12 media in a 37^oC water bath.

12) Aspirate media with DNA/ Lipofectamine 2000 solution mix from each plate and add 20 ml of DMEM-F12 media per 15 cm plate.

13) Keep cells in a humidified incubator (37^oC, 5% CO₂) for 24 hours.

Day 3:

1) Check transfected cells under light microscope. Successfully transfected cells should show a slight change in morphology, where cells look a little rounded.

2) Collect the supernatants from 2 x 15 cm dishes (40 ml in total) into a 50 ml falcon tube.

3) Spin down at 1000 rpm for 5 minutes at 25^oC to remove dead cells.

4) Collect the supernatant and subject supernatant to filtration using a 50 ml syringe attached to a 0.45 µm syringe filter. Collect the filtered supernatant in a new 50 ml falcon tube.

5) The filtered supernatant contains the desired virus. To concentrate the virus, transfer 10 to 15 ml of the filtered supernatant into a 100K Amicon Ultra-15 concentrator. Spin down at 4000 rpm for 30 minutes at 4^oC.

6) Once concentrated, approximately 100 µl of concentrated virus can be recovered per 100K Amicon Ultra-15 concentrator. Discard flow-through. Pool all concentrated virus into one 1.5 ml eppendorf tube and mix gently.

7) Aliquot 50 µl of virus per 1.5 ml eppendorf tube and flash freeze either with liquid nitrogen or isopropanol in dry ice.

8) Store virus at -80^oC until further use. Thawed viruses cannot be reused.

9) 70 µl of virus can be used to infect rat hippocampal/cortical neurons (35,000 cells/well) of one well of a 12-well plate. At least 90% transduction rate (assessed by EGFP expression) is expected.