Cloning

Cloning - May

Cloning - June

Cloning - July

Cloning - August

Cloning - September

Cloning - October

Viral Vectors

Viral Vectors - May

2014/05/21

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 using polyethylenimine.

• remove medium and refill with 5 ml new completed growth medium (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 was collected after 48 h (refilled with 5 ml DMEM) as well as 72 h.

2014/05/25

Transduction mouse cells

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

• 500 µl of supernatant was removed
• 1 µl Polybrene was added (10mg/ml)
• 500 µl virus supernatant was added (sterile 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.

Viral Vectors - June

2014/06/20

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 completed growth medium and centrifuged at 900 rpm for 2 min
• medium was removed and refilled with 10 ml warm completed growth medium
• cells were seeded on 100 mm plates

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. NIH 3T3 cells grow very fast; therefore we have tested the optimal seeding cell number to obtain 60% cell density on the next day. Results indicate that the optimal cell number is 1 &ndash 1.5x10^5 cells per well ( = 0.5 – 0.75 cells/ml)

2014/06/22

Transfection/ Virus production

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

2014/06/24

Transfection/ Virus production

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

2014/06/27

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 viral transduction with MuLV IRES EGFP, medium change after 16 h.

2014/06/27

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 (500µl viral supernatant, 500µl completed growth medium + 1µl Polybrene/ml) harvested at different time points. Results indicate that there was no difference between older and newer virus; best results were given with an infection time of 2 x 8 hours.

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. However, we found out that cells were death after centrifugation.

2014/06/30

Transfection CHO cells with receptor

CHO cells were transfected with the receptor (for later transduction). Medium was removed and filled with 2 ml new medium per well. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

Transfection/ Virusproduction

Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21).

Viral Vectors - July

2014/07/03

Transfection CHO cells with receptor

CHO cells were transfected with the receptor (for later transduction). Medium was removed and filled with 2 ml new medium per well. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C. This time there were no results du to high density of cells during transduction.

Freezing (cryopreservation) of eukaryotic cells

Phoenix cells were frozen at -80°C.

• removal of medium and washing with cold PBS
• addition of 1 ml 0,05% Trypsin per plate, incubation for 1-2 min)
• stopping of reaction with 5 ml DMEM (with FCS)
• centrifugation (5 min, 900 rpm)
• removal of supernatant and resuspension in 2 ml FCS (+10% DMSO)
• quick transfer in steril cryotube (1ml per tube) and quick freezing in -80°C

2014/07/06

Transfection CHO cells with receptor

CHO cells were seeded on cover slips and transfected with the receptor (for later transduction). Due to the fact that cells must be in growth phase during transduction with virus the cell density was set to 40%. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

Viral Vectors - August

Viral Vectors - September

Viral Vectors - October

Light-System

Light System - May

Light System - June

Light System - July

Light System - August

Light System - September

Light System - October

The Combination

The-Combination-May

The-Combination-June

The-Combination-July

The-Combination-August

The-Combination-September

The-Combination-October