Cloning

Cloning-May

Cloning-June

Cloning-July

Cloning-August

Cloning-September

Cloning-October

Virale Vectoren

Virale-Vectoren-May

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.

Virale-Vectoren-June

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.

• cells were dead after centrifugation

30.6.14

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/ Virus production

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

Virale-Vectoren-July

3.7.14

Transfection CHO cells with receptor

Cho cells were transfected with the receptor (for later transduction). Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

• cell density was to high for transduction

Stocking eukaryotic cells

Phoenix cells were stocked (-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

6.7.14

Transfection CHO cells with receptor

Cho cells (cover slips in each well) were transfected with the receptor (for later transduction). Cell density 40% due to the fact that cells must be in growth phase during transduction with virus. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

8.7.14

Transfection/ Virus production

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

10.7.14

Transfection CHO cells with receptor

Cho cells were transfected with the receptor (for later transduction). . Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP directly after transfection (after medium changing).

• no results

Fixation cells on cover slips

CHO cells (transfected with SLC7a1; 2014/07/04) were fixed on cover slips

• medium was removed and cells were washed with PBS
• appropriate amount of 4% PFA/PBS was added (200µl on 24W) and incubated for 10 min on ice
• PFA was removed and plate was washed with PBS
• cover slips were fixed with Mowiol on slides

11.7.14

Transfection HEK cells with receptor

HEK 293 cells were transfected with the receptor (for later transduction). Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

14.7.14

Transfection/ Virus production

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

17.7.14

Transduction mouse cells

Different volumina of virus supernatant were added to mouse cells (on 24W plate, 70% density) and analysed by FACS, Microscopy and Western Blot after 48 h.

green: anaysis by FACS.

yellow: fixation with PFA on cover slips

• removal of medium
• washing with cold PBS
• adding of 400 µl PFA and incubation for 10 min on ice, another 10 min at RT
• incubation of cover slips (pincette) for 10 sec in DAPI solution (1:5000 in water)
• washing in water
• fixation with mowiol

1. no results (better use poly-lysine for better grip of cells on cover slip)

blue: preparation for Western Blot via Ripa Lysis (as positive control for anti-CAT1 antibody)

• removal of medium
• washing with ice cold PBS
• addition of 100 µl Ripa Buffer (completed with Phosphatase-Inhibitor-Mix)
• incubation 10 min on ice
• removal of cells with tip and transfer into Eppendorf tube
• incubation for 10 min on ice
• centrifugation for 5 min 10000 x g
• transfer of 60 µl supernatant in new tube
• addition of 15 µl 5 x SDS loading dye (with Mercaptoethanol)
• cooking for 10 min at 95°C or for 15 min at 72°C
• freezing at -24°C

Transfection CHO with Receptor

Two different kinds of CHO cells (K1 and DMEM cells) were compared. Cells were transfected with the receptor for analysis with Western Blot. Both kinds of CHO cells were transfected with pMIG IRES EGFP and analysed with FACS to see which cells are better for transfection. Medium was changed after 5 h. Cells were incubated for 24 h at 37°C.

21.7.14

Transfection/ Virus production

Phoenix cells were transfected with pMIG IRES EGFP and pM

31.7.14

Improvement of Transduction

Transduction of NIH 3T3 cells with two different viral supernatants via three different methods

1. 2 µl Polybrene adding directly to 1 ml DMEM on cells and adding 1 ml viral supernatant afterwards
2. addition of 1 µl Polybrene to 1 ml viral supernatant and addition of the mixture to 1 ml DMEM on the cells
3. addition of 2 µl Polybrene to 1 ml viral supernatant and addition of the mixture to 1 ml DMEM on the cell

Transduction of NIH3T3 cells with two different virus with either 2 or 1 µl Polybrene

1. 1 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant
2. 2 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant

Centrifugation at 37°C for 45 min at 400 RPM

àcentrifugation is not good for cells

Virale-Vectoren-August

1.8.14

Erzeugung einer GFP Maus-Zelllinie

Um zu überprüfen , ob der MuLV stabil Gene in Zellen einbringen kann, wird eine stabile GFP Maus-Zelllinie erzeugt.

Eine 100mm plate wird mit 3 ml Virussupernatant (pMIG IRES EGFP) transduziert, Zelllinie wird ganz normal weiter gesplittet.

Transduction mouse cells (dilution)

NIH3T3 cells were transduced with MuLV IRES EGFP (different volumina of viral supernatants, up to 1 ml with DMEM), FACS analysis after 48 h

14.07.14

Western Blot

For two gels the following mixes were prepared; Stacking gel:

• 25 ml Acrylamide
• 15 ml 0.375 M Tris/HCl, ph 6.8
• 5 ml water
• 5 µl 10% SDS
• 250 µl APS
• 5 µl TEMED

Separation gel:

• 25 ml Acrylamide
• 05 ml 1 M Tris/HCl, pH 8.8
• 2 ml water
• 3 ml 50% sucrose
• 5 µl 10% SDS
• 235 µl APS
• 5 µl TEMED