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.

https://static.igem.org/mediawiki/2014/6/66/Freiburg2014-05-25_bild_1.png

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

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