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Team:Freiburg/Content/Notebook/Labjournal
From 2014.igem.org
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.
Phoenix cells transfected with pMIG IRES EGFP one day after transfection.
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.
NIH 3T3 cells were transduced twice with MuLV IRES EGFP for 6h. Picture was made after 48 h of incubation at 37°C.
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)
3 Phoenix cells transfected with pMIG IRES EGFP.
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.
(left) Cho cells without receptor were transduced with MuLV IRES EGFP ,(right) CHO cells transfected with SLC7a1 and transduced with MuLV IRES EGFP (24h after transfection). Analyses with flow cytometry indicates that 5% of cells were transfected (transfection control) and 2% of cells transfected with the receptor were infected with MuLV.
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.
CHO cells were transduced with MuLV IRES EGFP 24 h after transfection with SLC7A1.
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.
CHO cells transfected with SLC7a1 and transduction with MuLV IRES EGFP 24 h after transfection (A) experimental scheme. CHO cells were transduced with MuLV IRES EGFP 24 h after transfection with SLC7a1. (B) Transduced CHO cells expressing EGFP.
2014/07/08
Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21)
FACS results of pMIG IRES EGFP transfected Phoenix cells. Phoenix cells were transfected with pMIG IRES EGFP for production of viral particles. They were anaylsed by flow cytometry after three days of virus production (middle and upper pictures); negative control (upper picture).
2014/07/10
Transfection of CHO cells with receptor
CHO cells were transfected with the receptor (for later transduction). Medium was changed after 5 h and cells were transduced with MuLV IRES EGFP. This experiment gave no results.
2014/07/10
Fixation of 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
2014/07/11
Transfection of 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.
FACS results of pMIG IRES EGFP transfected Phoenix cells. Phoenix cells were transfected with pMIG IRES EGFP for production of viral particles. They were anaylsed by flow cytometry after three days of virus production (middle and upper pictures); negative control (upper picture).
2014/07/17
Transduction of mouse cells
Different volumes of virus supernatant were added to mouse cells (on 24W plate, 70% density) and analyzed by FACS (green), Microscopy (yellow) and Western Blot (blue) after 48 h.
green: anaylsis with flow cytometry
NIH cells transduced with MuLV IRES EGFP (left: 0,5 ml Virus + 0,5 ml DMEM; middle: 0,75 ml Virus + 0,25 ml DMEM; right: 1 ml Virus + 0 ml DMEM). Pictures made after 48 h.
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 for 10 sec in DAPI solution (1:5000 in water)
- Washing in water
- Mounting with Mowiol on slides
Cells detach from the cover slip, therefore a coating is necessary e.g. with Poly-L-Lysine 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 β-Mercaptoethanol)
- Cooking for 10 min at 95°C or for 15 min at 72°C
- Freezing at -24°C
Transfection of CHO cells with receptor
CHO cells growing in completed HTS medium (K1) were compared to CHO cells growing in completed DMEM medium. Cells were transfected with the receptor. Afterwards both kinds of CHO cells were infected with MuLV IRES EGFP and analyzyd using flow cytometry investigate which cells are better for transfection and transduction. Medium was changed after 5 h. Cells were incubated for 24 h at 37°C.
FACS results of CHO HTS (left) and CHO DMEM (right) cells transfected the receptor and transduced with MuLV EGFP.
2014/07/21
Transfection/ Virus production
Phoenix cells were transfected with pMIG IRES EGFP and pMIG EGFP(protocol: 2014/05/21)
FACS results of pMIG IRES EGFP transfected Phoenix cells. Phoenix cells were transfected with pMIG IRES EGFP for production of viral particles. They were anaylsed by flow cytometry after three days of virus production (middle and upper pictures); negative control (upper picture).
2014/07/31
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 cells
- Incubation for 48 h at 37°C
NIH3T3 cells were transduced with MuLV EGFP (upper pictures) and with MuLV IRES EGFP (lower pictures) left: transduction method 1; middle: transduction method 2; right: transduction method 3
Transduction of NIH3T3 cells with two different viral supernatants (MuLV IRES EGFP or MuLV EGFP) with either 1 or 2 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
- 3. 2µl Polybrene was added to 1 ml viral supernatant; mixture was added to 1 ml DMEM
Centrifugation at 37°C for 45 min at 400 RPM. Results indicate that cells do not like to be centrifuged.
NIH3T3 cells transduced with MuLV EGFP (A + B) and MuLV IRES EGFP (C + D) either with 1 µl (A + C) or 2 µl (B + D) Polybrene and centrifuged 45 min at 37°C for 400 RPM
Testing difference in transfection efficiency of Phoenix and HEK cells/ negative control for MuLV
Phoenix cells and HEK cells were tested for there transfection capacity and compared. In addition, it was tested that the virus cannot infect Phoenix nor HEK cells. So both kinds of cells were transfected with PHB308 (mCherry, 3 µg/well) and in parallel transduced with MuLV IRES EGFP (1 ml/well + 2 µl Polybrene).
HEK cells (A + B): transduced with MuLV IRES EGFP (A); negative control (B); Phoenix cells (C + D): transduced with MuLV IRES EGFP (C); negative control (D); HEK cells (E) and (F) Phoenix cells transfected with PHB308 and analysed after 24 h.
Comparison of transfection capacity of HEK 293 cells (red) and Phoenix cells (blue). Both cell lines were transfected with 3 µg PHB308 (mCherry) per well (6W plate) and analysed via FACS after 24 h.
Viral Vectors - August
2014/08/01
Generation of a GFP mouse cell line
For testing whether MuLV can stable transfer genes into cells, a stable mouse cell line using this virus was generated.
Therefore two 100mm plates were transduced with 3 ml virus supernatant (MuLV IRES EGFP) and splitted as usual. Cells were sorted with a cell sorter. Analysis via FACS happened before and after sorting. The analysis was repeated after several rounds of splitting.
NIH3T3 cells transduced with MuLV IRES EGFP and sorted. Negative control (left), before sorting (middle), after sorting (right).
2014/08/03
Testing different transfection methods with different cells
For optimizing transfection in different cell lines transfection methods and different concentrations of the transfection mixtures were compared. The experiment was done with mouse cells (NIH3T3), hamster cells (CHO) and human cells (Phoenix). As transfection reagents lipofectamin and PEI were used in different concentrations.
NIH3T3 cells transduced with MuLV IRES EGFP and sorted. Negative control (left), before sorting (middle), after sorting (right).
Transfection with Lipofectamin (for 3 wells):
- (solutions A) 50 µl OptMEM was mixed with either:
- 1. 1 µl Lipofectamin + 1,5 µl PHB308
- 2. 2,5 µl Lipofectamin + 1,5 µl PHB308 or
- 3. 4 µl Lipofectamin + 1,5 µl PHB308
- incubation for 25 min at RT
- (solution B) 150 µl OptiMEM was mixed with 1,5 µl PHB308 (2,5 µg/µl) and 4 µl Plut Reagent
- incubation for 15 min
- solutions A (1-3) were then mixed with 50 µl of solution B and incubated for 5 min at RT
- 100 µl of transfection solution were added to each well
- no medium changing, incubation at 37°C for 24 h
Transfection with PEI (for 1 well):
- 0,2 µl PHB308 was mixed with 40 µl OptiMEM and DNA, for each well another concentration of DNA was added:
- 1. 1,5 µl PEI
- 2. 3 µl PEI
- 3. 5 µl PEI
- Incubation for (optimal) 10,8 min
- addition of 40 µL solution to each well
- medium changing after 5 h of incubation at 37°C, incubation for 24 h at 37°C
Pictures and FACS data of different transfection methods with different kinds of cells. A: Phoenix cells transfected with PHB308 (mCherry) via 1 µl (left), 2,5 µl (middle) and 4 µl Lipofectamin; B: Phoenix cells transfected with PHB308 (mCherry) via 1,5 µl (left), 3 µl (middle) and 5 µl Pei; C: Cho cells transfected with PHB308 (mCherry) via 1 µl (left), 2,5 µl (middle) and 4 µl Lipofectamin; D: Cho cells transfected with PHB308 (mCherry) via 1,5 µl (left), 3 µl (middle) and 5 µl Pei; E: NIH3T3 cells transfected with PHB308 (mCherry) via 1 µl (left), 2,5 µl (middle) and 4 µl Lipofectamin; F: NIH3T3 cells transfected with PHB308 (mCherry) via 1,5 µl (left), 3 µl (middle) and 5 µl Pei.
2014/08/06
Transfection of HEK cells with receptor
HEK293 were transfected with the receptor plus mCherry to see, if the virus infect only cells expressing the receptor (the two plasmids should infect same cells). In parallel cells were transfected with the blue light and the red light positive control (PMZ422 and PSAM200) and the receptor inducible by the light systems. After 24 h cells (expressing the receptor) were infected with MuLV IRES EGFP.
HEK 293 tranfected with PHB308 (mCherry) and the receptor. Transduction with MuLV after 24 h of incubation. Pictures were taken two days post transfection.
Transfection HEK and Pheonix cells with receptor (transfection while seeding)
To avoid a too high cell density for transduction cells were transfected while seeding. The PEI transfection mix was given to the HEK or Phoenix cell suspension (1,5 x 105 c/ml, 100 µl mix per 1 ml cells) and seeded on a 12W plate. Results indicated that PEI was too toxic for cells during this method.
2014/08/09
Virus dilution
Virus supernatant was diluted with fresh DMEM before transduction. Cells were analyzed after 48 h.
- 0,5 ml virus + 0,5 ml DMEM
- 0,4 ml virus + 0,6 ml DMEM
- 0,3 ml virus + 0,7 ml DMEM
- 0,2 ml virus + 0,8 ml DMEM
- 0,1 ml virus + 0, 9 ml DMEM
NIH3t3 cells transduced with MuLV IRES EGFP in different dilutions (upper from left to right: 5/10, 4/10, 3/10, 2/10, 1/10 virus supernatant. Pictures were made after 48 h of incubation at 37°C.
NIH3t3 cells transduced with diluted MuLV IRES EGFP and analysed by FACS after 48 h, red: 1/10, blue: 2/10, orange: 3/10, green: 4/10, dark green: 5/10 dilution.
Viral Vectors - September
2014/09/01
QR-code on 96W plate
HEK cells were transfected with the blue light system with receptor and seeded on a 96W plate for pattern generation.
- Transfection of HEK293 in suspension (2 x 105 cells/ml, 100µl transfection mix/ml) with PKM292, PKM297 and PKM084 (1,5 µg DNA/ml cell suspension)
- Incubation for 5 h at 37°C and seeding on a 96W plate (with photo mask) afterwards (120µl/well)
- After 24 h of incubation at 37°C cells were illuminated with blue light for 5 h.
- Again after 24 h of incubation a SEAP assay was made directly in the plate:
- 30 min incubation of plate at 65°C
- addition of 100µl SEAP buffer into each well (ca. 90µl medium was left in each well)
- before measurement 20 µl substrate was added
left: Supernatants of HEK293 cells transfected with PKM292, PKM297 and PKM084 24 h after illumination with blue light. For visualizing of SEAP a SEAP assay was made (2014-09-03 23.55.54), right: same supernatants with with colour switch that was made after taking picture.
2014/09/03
Experiment: receptor life time after splitting
Two 6W of HEK293 cells were transfected with rz006 (receptor labled with mCherry and HA-tag). After each round of splitting cells were analysed by Western blot. Therefore each round one well was splitted 1:2 on two new wells and one well was lysed by Ripa lysis. Afterwards, each lysed sample was frozen in -20°C until use.
Two new cells lines
New cells lines were thawed:
- MCF-7 human breast cancer cells
- A-547 human lung epithel carcinoma
Rezeptorexpression time
HEK293 cells on 16 x 35mm plates were transfected with rz006 (receptor labeled with mCherry and HA-tag). At several time points after transfection cells were lysed with RIPA buffer and analyzed with Western Blot. Samples were frozen at -20°C until analysis.
Western blot of ripa lysed HEK293 cells expressing rz006 (labled with HA-tag). Samples were taken after several time points after transfection.
2014/09/26
Transfection/ Virus production (stained virus)
Phoenix cells were stained with DiD, a membrane staining dye.
- Resuspension of 2 µl dye (5mM) in 5 ml OptiMEM
- Removing of medium from five 100mm Pheonix plates and addition of 1 ml staining solution per well
- Incubation for 15 min at 37°C
- Washing of plates two times with DMEM (incubation time between each washing step was at least 10 min
- Transfection of cells with pMIG-mKO, pMIG-BFP2, pMIG-EGFP and pMIG-mKate
- Medium changing after 24 h of incubation at 37°C and refill with 4 ml new DMEM
- Harvest after 48 h and 72 h post transfection
2014/09/28
Virusintegration time in mouse cells
NIH3t3 cells (mouse fibroblasts) were infected with MuLV IRES EGFP at different time points. Directly after the latest infection the cells were splitted on new plates. After 48 h cells were analyzed via FACS.
QR-code on 24W plate (with virus)
For testing the functionality of our system and the functionality of the virus with SEAP, cells were transfected with the blue light system plus light inducible receptor (labled with mCherry) and later infected with MuLV SEAP or MuLV CMV SEAP. Both viruses were compared. In addition, cells were infected with MuLV EGFP as control. Several negative controls were added as well as unilluminated cells expressing the light system for testing leaky expression. Cells were incubated in dark.
- Transfection of cells with eather the blue light system plus receptor (PKM292, PKM297, ls003) or the receptor (rz006 p2a mCherry).
- Cells were transfected via PEI in suspension (3 x 10^5 cells/ml, 100 µl transfection mix/ml, 1,5 µg DNA/ml). Cells were incubated for
- 5 h at 37°C before centrifugation (900 RPM, 3 min, 24°C). Cells were resuspended with fresh DMEM to a final concentration of 1,5 x 10^5 cells/ml) and seeded (0,5 ml/well).
- Cells were incubated for 24 h at 37°C and the illuminated with blue light for 1 h (not the dark control).
- Cells were infected with 250 µl virus supernatant (+0,5 µl Polybrene) per well. Medium was changed after 4 h of incubation at 37°C.
- After 24 h and after 48 h of incubation 200 µl supernatant was taken and used for SEAP assay as described before.
