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From 2014.igem.org

Virale Vectoren

21.05.14

Transfection/ Virus production

For virus production Phoenix cells (producer cell line) were splitted (well separated) on 100mm plates. At 70% cell density cells were transfected via PEI transfection.

• remove medium and refill with 5 ml new DMEM
• 600 µl transfection mastermix was prepared (8 µg pMIG IRES EGFP, 24 µl PEI, rest OptiMEM)
• mastermix was incubated 15 min and carefully drop on the plates

Plates were incubated at 37°C. The supernatant after 24 was removed and refilled with 5 ml new DMEM, the supernatant after 48 h was collected (refilled with 5 ml DMEM) as well as the supernatant after 72 h.

25.4.14

Transduction mouse cells

NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP.

• 500 µl of supernatant was removed
• 1 µl Polyprene was added (10mg/ml)
• 500 µl virus supernatant was added (steril filtered)
• incubation at 37°C for 6h
• cell supernatant was replaced with fresh DMEM
• transduction was repeated once

Pictures could be made after 48 h of incubation.

20.6.14

Thawing of eukaryotic cells

New Phoenix cell stocks were thawed:

• cryotube was thawed at 37°C water bath until almost defrosted
• stock was filled in 9 ml warm DMEM and centrifuged at 900 rpm for 2 min
• medium was removed and refilled with 10 ml warm DMEM
• cells were seeded on 100 mm plate

Testing optimal cell density of mouse fibroblasts

NIH 3T3 have a really fast growth so that we tested the optimal cell number for seeding NIH 3T3 for having around 60% cell density on the next day.

• incubation for 24 h at 37°C

àoptimal cell number is 1 - 1,5x10^5 cells per well ( = 0,5 – 0,75 cells/ml)

22.6.14

Transfection/ Virus production

Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (2 x 100mm plate)

24.6.14

Transfection/ Virus production

Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (5 x 100mm plate)

27.6.14

Thawing new HEK 293 cells

(protocol: 2014/06/20)

Transfection CHO cells with receptor

Transfection of CHO cells with SLC7a1 (for later transduction with virus). Medium was changed after 5 h. Cells were incubated for 24 h before transduced with Mulv IRES EGFP, medium change after 16 h.

FACS analysis: CHO + SLC7a1 transduced: 2%, Transfection control: 5%

29.6.14

Transduction mouse cells (different incubation times)

NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP and incubated for 8, 16, 24 and 2 x 8 hours. Virus was taken from different supernatants (an older one and a newer one) to see, if it makes any difference. Cells were infected with supernatant harvested at different time points.

• 500 µl virus supernatant was added to 500 µl DMEM per well + 1 µl Polybrene

1. there was no difference between older and newer virus, best results gave 2 x 8 h incubation

For testing, if centrifugation brings better transduction efficiencies, mouse cells were infected with the different viral supernatants and centrifuged for 45 min, 1800 rpm, 32°C. In two wells it was tested if the double amount of Polybrene brings better transduction efficiencies.