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Cellculture

This subpage is for cellculture, the structure of each experiment is following:

Date of first action, title of the experiment

The motivation
This short introduction provides an inside into our thoughts for this experiment.
The plan
In this section, the design of the experiment is explained.
The results
Interpretation and discussion of our results.

14.6 Seeding AAV293 for transfection

The motivation
We want to create viral stocks with the stratagene R/C, the gene of interest is mVenus. This is a qualitative control.

The plan
AAV 293 and HT1080 Cells have been splitted and plated out on 10 cm dishes for transfektion. The Cells were accounted by using the Neubauer-Meteringchamber. After harvesting by following the standard protocol the pallet have been resuspendiated in 15ml of DTT medium. 2,5µl of this cell suspension have been mixed with 47.5µl of trypan blue.

We counted 12,5x 10^6 cells/ml for the AAV293 cell line and 10x10^6 cells /ml for the HT1080 cell line.

The AAV 293 cells have been plated out on four dishes with 250µl of the cell suspension, on one plated with 1ml and on another dish with 1.5ml. note that that the date is wrong on the plates and the flasks! Cells have been already plated out on the 14th. of June.

We also seated two flasks one for each cell line. Therefor we used 1ml of the HT1080 cell suspension and two ml of the the AAV293 cells.
1)

• pAAV_iGEM_mVenus_YFP (glycerol stock B34, P39) P39 has the correct sequence (confirmed)
• pHelper
• pAAV_RC

2)

• pAAV_iGEM_mVenus_YFP (glycerol stock B33, P38) P38 (we dont know if P39 has the correct sequence!)was used because the amount of P39 was not sufficient enough for four Transfections!
• pHelper
• pAAV_RC

Plasmid concentrations:
pAAV_RC: 1 µg/µl
pHelper: 280 ng/µl
pAAV_iGEM_mVenus_YFP, P39: 180,83 ng/µl
pAAV_iGEM_mVenus_YFP, P38: 179,75 ng/µl

Cellculture clone 1 and 2 were transfected with P39. Cellculture clone 3 and 4 were transfected with P38.

We could only pipet 3,3 µg (instead of 10 µg) from each of the 3 plasmids into the 15 ml falcons due to insufficient amount of plasmid.

Adrian tried to examine the precipation of the CaCl2+ DNA Clusters. We have to optimize the pH of our 2xHBS at the moment it is 11.12.
The viral stocks are harvested according to standard protocol.
The results

Results of transduction, ~ 1% of the HT1080 showed expression of mVenus.

Bright light image 1	Fluoresce microscopy image 1
Bright light image 1	Fluoresce microscopy image 1

According to the stratagene protocol, transduction efficencies about ~80% are achievable. We performed every step under guidance, so probably something is wrong with the buffers, cells or even the standard protocol. The next step is to repeat this transfection.

19.6 Seeding AAV293 for second transfection according to standard protocol

The motivation
We want to repeat the last transfection to exclude faults in practical
The plan
Six transfections were performed according to the standart protocol: Media:Freiburg10_Transfection_protocoll.pdf
Three 10 cm cellcluture dishes with 293 cells were transfected with 10 µg DNA (3,33 µg each plasmid) and the other three 10 cm cellculture dishes with 30 µg DNA (10 µg DNA each plasmid).

Plasmids:

• pAAV_iGEM-MCS_mVenus (P41), 507 ng/µl
• pAAV_RC , 1000 ng/µl
• pHelper , 280 ng/µl

• Transduction of 2x6 wells was successfully done (14.25)
• one six well was transduced with 10µg the other with 30µg DNA amount
  

The results

Results of transduction nr.2

10µg_unverd_2_(c1).JPG 48h post transduction	10µg_unverd_1_(c1).JPG 48h post transduction
30µg_unverd_2_(c1).JPG 48h post transduction	30µg_unverd_(c1).JPG 48h post transduction
30µg_unverd_4_(c1).JPG 48h post transduction	30µg unverd 3 (c1).JPG 48h post transduction
30µg_unverd_6_(c1).JPG 48h post transduction	30µg_unverd_5_(c1).JPG 48h post transduction, we changed the mVenus-yellow to pink, for the girls :)

The Transduction efficency is still quite low! We have to optimize the stratagene protocol!

30.6 Examination of different Transduction methods

The motivation
We want to rise the amount of transduced particels, instead of 500µl, 1000µl of the AAV-stock getting pipetted into each well. the second plate gets stimulated via UV-light to boost the expression of DNA-Repair mechanisms. This should enhance the second strand synthesis of our ssDNA-GOI => a rise of YFP Expression!

The plan

1. Plate I(A is up)

Transduction:

2. Plate II (A is up)

Transduction:

The results
All cells are dead. It was not possible to take pictures because the dead cells were all clumped up. Looks like we should not transduce 3x10^5 cells with 1000 µl (or more) of our viral solution because almost all cells died in all approaches and the medium indicator was yellow. So we can not say if resuspendig the cells with the viral soultion is a useful alteration of the standard protocol.

3.7 Seeding AAV293 for transfection with different plasmid amounts and harvesting methods

The motivation

We want to check if the amount of plasmids is the reason for the low transduction efficiency. Our main problem is that the flow cytometry (FACS) is not available, so the results have to be checked via fuorescence microscopy.

The plan

Transfection with P41 was performed according standart protocol. Two transfections where carried out with 10µg (3,33 µg each plasmid) DNA and the other two with 20µg DNA (6,66 µg each plasmid).
We want to investigate which of the following approaches yields a better transduction efficiency.

• Half of the transfected cells were exposed 3 cycles of thawing and freezing and then centrifuged (15 ml falcon, 2100G). The supernatant was transfered into a new 15 ml falcon and used for transduction.
• The other half was centrifuged at 300 G for 5min . The supernatant was transfered into another 15 ml falcon and the pellet was resuspended with 5 ml of DMEM. The content of these two falcons was also exposed to 3 cycles of thawing and freezing and then used for transduction.

• We have got 2 10 cm cellculture dishes with 10µg and 2 dishes with 20µg DNA used for transfection.

Approach with standart protocol (one dish with 10 and the other with 20µg Plasmids)(2100 G and 3 cycles of freezing and thawing)

Deviations from the standart protocol:

• The cells were centrifuged at 2100 G instead of 10.000 G
• The viruses were harvested after 44 hours
• 3 cycles of freezing and thawing

• used plasmids: 10µg, 20µg YFP
• we have 10µg and 20µg from standart protocol => SV
• we have 10µg and 20µg from standart protocol pellet => Pellet
• we have 10µg and 20µg from standart protocol suspension => Super
• amount of 500µl

1. Plate I(A is up)

2. Plate I(A is up)

3. Plate I(A is up)

The results

Results of transduction with different harvested viral particles

plate 1 A 1

plate 1 A 2

We only took some pictures from single cells, because there was no significant difference in fluorescence between each sample. The main conclusion of this experiment is, that we have to get the flow cytometry started.

4.7 Transfection with different amounts of plasmids

The motivation
We want to check if the mVenus expression/ transduction efficiency can be extended with higher amounts of DNA.
The plan
The Transfection was performed with 10, 18 and 24 µg per plasmid (mVenus, pHelper and R/C).
the harvest was performed according to standard protocol:

• 3 Plates got transduced

1. Plate I(A is up) completely 10 µg

2. Plate II(A is up)completely 18 µg

3. Plate III(A is up) completely 24 µg

The results
FACS analysis of different treated HT1080 cells was done (18 samples)

The first percentage refers to the living and fluorescent cells and the second percentage to the total amout of living cells.

The data are listed this way:

1. Plate I(A is up)

1= Plate 1 A1 : 0% YFP-positive Cells (there were only a few cells in this sample)
2= Plate 1 A2 : 27,7% YFP-positve Cells from 73,3%
3= Plate 1 A3 : 17,3% " from 63,1%
4= Plate 2 A1 : 32,9% " from 75,3%
5= Plate 2 A2 : 27,2% " from 76,5%
6= Plate 2 A3 : 30,7% " from 73,4%
7= Plate 3 A1 : 26,8% " from 76,0%
8= Plate 3 A2 : 30,5% " from 74,2%
9= Plate 3 A3 : 30,7% " from 75,2%
10= Plate 1 B1 : 6,25% " from 36,1%
11= Plate 1 B2 : 6,42% " from 68,4%
12= Plate 1 B3 : 0,05% " from 89,5%
13= Plate 2 B1 : 20,5% " from 72,5%
14= Plate 2 B2 : 15,7% " from 73,6%
15= Plate 2 B3 : 0,02% " from 85,4%
16= Plate 3 B1 : 19,5% " from 73,3%
17= Plate 3 B2 : 20,8% " from 75,7%
18= Plate 3 B3 : 0,07% " from 84,8%

The calculated value written into the table describes the fluorescent amount of cells from the living cells. Example: 1 A2 : 27,7% YFP-positve cells of 73,3% living cells : 27,7/73,3 = 0,378

Conclusions

• Higher amounts of Plasmids (24, 30µg) seems to have no obvious effect on YFP expression
• Theres no difference in YFP expression comparing resuspended samples to not resuspended samples
• The amount of dead HT cells is about 25%, it is not clear if it is either AAV induced or mechanic stress (Trypsin, washing procedure)

Next steps:

• does the amount of GOI (ng plasmids) has an effect on YFP expression
• compare transduced to non transduced cells (vitality), or in other words is the mechanic stress responsible for 25% dead cell ratio?
• comparison of transduction efficiency of different virus stock production routines

14.7 Seeding AAV293 for TKGMK constructs

The motivation
We want to create the first viral particles with TKGMK as gene of interest.
The plan

30µg Plasmid were used (pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30)
VIRUS no GOI :pHelper + pAAV_RC
VIRUS 1  :pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30 clone 1 : P54
VIRUS 2  :pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30 clone 2 : P55
VIRUS 3  :pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30 clone 3 : P56

The results
Unfortunately the stocks never were used.

7.8 Seeding AAV293 for TKGMK and mVenus stocks

The motivation First we want to create new stock of AAV without GOI, the next viral stock is with correct TK/GMK, the last stocks are with different YFP-amounts to check if the amount of GOI is a critical step for transduction effency (transgene expression)..

The plan

transduction was performed according to standard protocol.

Platte 1:

Platte 2:

Platte 3:

Platte 4:

Platte 5:

Platte 6:

Platte 7:

Platte 8:

The test for functionality for TK GMK was done with the Prodrug Cymeven ganciclovir 500mg from Roche. The final concentration was 0,05 mM in the wells. After two days pictures were taken.

The results

Death to HT1080

HT1080 with gancliclovir only	HT1080 with TK_GMK without ganciclovir
HT1080 with TK_GMK (300µl AAV stock) with ganciclovir	HT1080 with TK_GMK (600µl AAV stock) with ganciclovir
picture from the 40µg stock

As you can see, the TKGMK cell killing mechanism works! The samples with targeted mVenus constructs couldnt be checked because the FACS machine was not working. The fluorescence was checked with microscopy, the transduction efficiency is still low.

11.8 Transduction of HT1080 with no AAV, AAV without GOI and AAV with 10µg

The motivation: In the past, the percentage of living cells in FACS was very low (~ 70%) so we want to check if the AAV-induced stress is responsible for cellular death, or the detaching procedure (trypsin and mechanical stress).
The plan
Plate I

Plate II

Plate III

The results The YFP expression is still quite low. This was kind of anticipated, the success in this experiment is the fact,that the amount of living cells is at a very high level! (~90%). In future preparations of FACS samples use cold PBS!

14.8 Seeding different amounts of AAV293 for transfection

The motivation
We want to know which confluence of AAV293 cells is optimal for AAV production.

The plan
We seed different amounts of cells in 10 cm², and check the highest titer.

The cells were transfected with 40µg YFP, 3,3µg Rep/Cap and 3,3µg pHelper.

Transduction was performed according to standard protocol.
Plate 1 YFP: 200.000 cells per well

Plate 2 YFP: 200.000 cells per well

Plate 3 YFP: 200.000 cells per well

Plate 4 YFP: 200.000 cells per well

Plate 5 YFP: 200.000 cells per well

17.8 transduction with viral stocks 20 µg and 40 µg