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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)

3x10^5 cells	3x10^5 cells	3x10^5 cells
3x10^5 cells	3x10^5 cells	3x10^5 cells

Transduction:

1000 µl of Viral Stock DROPPED	1000 µl of Viral Stock gently resuspended	1500 µl of Viral Stock hard resuspending
1000 µl of Viral Stock DROPPED	1000 µl of Viral Stock gently resuspended	no Transduction

2. Plate II (A is up)

about 3x10^5 cells	only medium	only medium
about 3x10^5 cells	only medium	only medium

Transduction:

1500 µl of Viral Stock gently resuspended	no Transduction	no Transduction
1500 µl of Viral Stock gently resuspended	no Transduction	no 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)

10µg dropped Super	10µg dropped Super gently resuspending	10µg dropped Super gently resuspending
10µg Super gently resuspending Super	20µg dropped Super gently resuspending 1:10 dilution	control

2. Plate I(A is up)

10µg dropped Super	10µg dropped Super gently resuspending	10µg dropped Super gently resuspending
10µg Super gently resuspending Super	20µg dropped Super gently resuspending 1:10 dilution	control

3. Plate I(A is up)

10µg dropped Pellet	10µg dropped Pellet gently resuspending	10µg dropped Pellet gently resuspending
10µg Super gently resuspending Pellet	20µg dropped Pellet gently resuspending 1:10 dilution	control

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

300µl dropped	300µl resuspended	450µl resuspended
600µl dropped	600µl resuspended	control

2. Plate II(A is up)completely 18 µg

300µl dropped	300µl resuspended	450µl resuspended
600µl dropped	600µl resuspended	control

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

300µl dropped	300µl resuspended	450µl resuspended
600µl dropped	600µl resuspended	control

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)

A1	A2	A3
B1	B2	B3

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

Plate	pAAV_RC/µg	pHelper/µg	GOI/µg
1	3,3	3,3	no GOI
2	6,6	3,3	no GOI
3	3,3	3,3	3,3 TK_GMK clone1
4	3,3	3,3	3,3 TK_GMK clone1
5	3,3	3,3	3,3 TK_GMK clone2
6	3,3	3,3	3,3 YFP
7	3,3	3,3	10 YFP
8	3,3	3,3	20 YFP
9	3,3	3,3	40 YFP
10	3,3	3,3	60 YFP

transduction was performed according to standard protocol.

Platte 1:

	1	2	3
A	control, no cells	300µl virus 1	300µl virus 2
B	control, no virus	600µl virus 1	600µl virus 2

Platte 2:

	1	2	3
A	control, no cells	300µl virus 3	300µl virus 4
B	control, no virus	600µl virus 3	600µl virus 4

Platte 3:

	1	2	3
A	control, no cells	300µl virus 5	300µl virus 6
B	control, no virus	600µl virus 5	600µl virus 6

Platte 4:

	1	2	3
A	control, no cells	300µl virus 7	300µl virus 8
B	control, no virus	600µl virus 7	600µl virus 8

Platte 5:

	1	2	3
A	control, no cells	300µl virus 9	300µl virus 10
B	control, no virus	600µl virus 9	600µl virus 10

Platte 6:

	1	2	3
A	control, no cells	300µl virus 3	300µl virus 4
B	control, no virus	600µl virus 3	600µl virus 4

Platte 7:

	1	2	3
A	control, no cells	300µl virus 3	300µl virus 4
B	control, no virus	500µl virus 3	500µl virus 4

Platte 8:

	1	2	3
A	control, no cells	300µl virus 5	300µl virus 5
B	control, no virus	600µl virus 5	600µl virus 5

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

control, no cells	no GOI 150µl	10µg 500µl
control, no virus	no GOI 300µl	10µg 750µl

Plate II

control, no cells	no GOI 150µl	10µg 500µl
control, no virus	no GOI 300µl	10µg 750µl

Plate III

control, no cells	no GOI 150µl	10µg 500µl
control, no virus	no virus	10µg 750µl

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.

Plate	amount of cells
1	100 000
2	200 000
3	400 000
4	500 000
5	800 000
6	1 000 000
7	1 200 000
8	1 500 000
9	1 750 000
10	1 750 000

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

	1	2	3
A	control, no cells	165µl virus (1)	165µl virus (2)
B	control, no virus	165µl virus (1)	165µl virus (2)

Plate 2 YFP: 200.000 cells per well

	1	2	3
A	control, no cells	165µl virus (3)	165µl virus (4)
B	control, no virus	165µl virus (3)	165µl virus (4)

Plate 3 YFP: 200.000 cells per well

	1	2	3
A	control, no cells	165µl virus (5)	165µl virus (6)
B	control, no virus	165µl virus (5)	165µl virus (6)

Plate 4 YFP: 200.000 cells per well

	1	2	3
A	control, no cells	165µl virus (7)	165µl virus (8)
B	control, no virus	165µl virus (7)	165µl virus (8)

Plate 5 YFP: 200.000 cells per well

	1	2	3
A	control, no cells	165µl virus (9)	165µl virus (10)
B	control, no virus	165µl virus (9)	165µl virus (10)

17.8 transduction with viral stocks 20 µg and 40 µg

1. plate 50.000 cells, YFP

A	control no cells	300 µl virus (40 µg YFP)	300 µl virus (20 µg YFP)
B	control no virus	500µl P38 (pAAV2_mVenus)	1000µl P38 (pAAV2_mVenus)

2. plate 50.000 cells, YFP

A	control no cells	300 µl virus (40 µg YFP)	300 µl virus (20 µg YFP)
B	control no virus	500µl P38 (pAAV2_mVenus)	1000µl P38 (pAAV2_mVenus)

3. plate 100.000 cells, YFP

A	control no cells	300 µl virus (40 µg YFP)	300 µl virus (20 µg YFP)
B	control no virus	500µl P38 (pAAV2_mVenus)	1000µl P38 (pAAV2_mVenus)

4. plate 100.000 cells, YFP

A	control no cells	300 µl virus (40 µg YFP)	300 µl virus (20 µg YFP)
B	control no virus	500µl P38 (pAAV2_mVenus)	1000µl P38 (pAAV2_mVenus)

5. plate 100.000 cells TKGMK

A	control no cells	150 µl virus (TKGMK clone 1)	180 µl virus (TKGMK clone 1)
B	control no virus	200 µl (TKGMK clone2)	200µl (no GOI)

16.8 Seeding AAV293 for transfection with new R/C

The motivation
We want examine the functionality of different R/C constructs and the new eGFP gene of interest. This transfection was performed with new cells which were thawed.

The plan

2x10⁶ cells were seeded (instead of 3x10⁶) --> hopefully better virus-production
used plasmids:
- P50 c= 429 ng/µl (RC)
- P64 c= 500 ng/µl (pHelper)
- P229 c= 162 ng/µl (GOI=YFP)
- P228 c= 540 ng/µl (GOI=eGFP)
- P158a c= 1800ng/µl (RC mutant)
- P158a c= 1770ng/µl (RC mutant)

The transfection was performed according to standard protocol.

1. plate: Lipo-Transfection
2. plate: Lipo-Transfection
3. plate: 3,3 µg (RC), 3,3 µg (pHelper), 3,3 µg (YFP)
4. plate: 10 µg (RC), 10 µg (pHelper), 3,3 µg (YFP)
5. plate: 10 µg (RC mutant P158a), 10 µg (pHelper), 3,3 µg (YFP)
6. plate: 10 µg (RC mutant P158b), 10 µg (pHelper), 3,3 µg (YFP)
7. plate: 3,3 µg (RC mutant P158a), 3,3 µg (pHelper), 3,3 µg (YFP)
8. plate: 3,3 µg (RC mutant P158b), 3,3 µg (pHelper), 3,3 µg (YFP)
9. plate: 10 µg (RC), 10 µg (pHelper), 3,3 µg (eGFP)
10. plate: 10 µg (RC), 10 µg (pHelper), 10 µg (eGFP)
11. plate: 10 µg (RC), 10 µg (pHelper), 20 µg (eGFP)
12. plate: 10 µg (RC), 10 µg (pHelper), 40 µg (eGFP)

The results

As you can see the eGFP constructs very well. And even the transduction efficiency of the standard R/C is doubled.

20.8

The Motivation
Seeding HT1080 for transfection with viral stocks from 18.8
The Plan
We harvest four T75 flasks and seed them into 6er dishes (200.000 cells in each well)
the Results

The Facs data at 22.8

19.8 Seeding AAv293 for transfection with different HBS buffers

The Motivation
We want to try different 2xHBS Buffers (pH) and their influence on transfection efficiency!

The Plan

We try 5 different 2xHBS buffers:

pH: 7.06
pH: 7.08
pH: 7.10
pH: 7.12
pH: 7.14

Used Plasmids: Standard protocol: 10µg of each plasmid

GOI: mVENUS => P262, conc:1148ng/µl; used amount: 8,71µl
GOI: TKGMK => P82b, conc:1500ng/µl; used amount: 6,67µl
pHELPER => P64, conc:503ng/µl; used amount: 20µl
RepCap => 10.8: 1348ng/µl; used amount: 7,41µl

Eight new viral stocks were made with pHelper:20µl and RepCap:7,41µl each:

mVenus:8,71µl; pH of 2xHBS:7,06
mVenus:8,71µl; pH of 2xHBS:7,08
mVenus:8,71µl; pH of 2xHBS:7,10
mVenus:8,71µl; pH of 2xHBS:7,12
mVenus:8,71µl; pH of 2xHBS:7,14
TKGMK:6,67µl; pH of 2xHBS:7,08
TKGMK:6,67µl; pH of 2xHBS:7,10
TKGMK:6,67µl; pH of 2xHBS:7,12

Transduction of three 6-well plates:

Plate 1 YFP: 150.000 cells per well

	1	2	3
A	control, no cells	500µl virus (1)	500µl virus (2)
B	control, no virus	500µl virus (1)	500µl virus (2)

Plate 2 YFP: 150.000 cells per well

	1	2	3
A	control, no cells	500µl virus (3)	500µl virus (4)
B	control, no virus	500µl virus (3)	500µl virus (4)

Plate 3 YFP: 200.000 cells per well

	1	2	3
A	control, no cells	500µl virus (5)	500µl virus (9)
B	control, no virus	500µl virus (9)	500µl virus (5)

(1)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,06
(2)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,08
(3)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,10
(4)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,12
(5)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,14
(9)= 10µg RC, 10µg pHelper, 3,3µg GOI (eGFP)

the Results
AAV-Harvesting is at 24.8, same day Transduction and FACS will be done at 26.8

As you can see the 2xHBS with pH 4.10 performed best, in future experiments this buffer will be used.

XXXXXXXXXXXXXXXXXXXXXXXXXX

Checking transduction efficiency via fluorescence microscopy

the motivation

The transduction efficiency from the viral stocks which were created with different amounts of AAV293 was checked.

The plan

plate 1:

stock 1 (with 100.000 cells)
stock 2 (with 200.000 cells)

plate 2:

stock 3 (with 400.000 cells)
stock 4 (with 500.000 cells)

plate 3:

stock 5 (with 800.000 cells)
stock 6 (with 1.000.000 cells)

plate 4:

stock 7 (with 1.200.000 cells)
stock 8 (with 1.500.000 cells)

plate 5:

stock 9 (with 1.750.000 cells)
stock 10 (with 1.750.000 cells)

The results

the optimal confluence of AAv293 cells is between 100 000 and 800 000 cells. This will be the field of investigation in the next experiments

25.8: Transfection of AAV293 with different amounts of cells, and two plasmid concentrations

The motivation

We want to define the final amount of AAV293 per 10 cm² for optimal AAV-Production

The plan

100.000
100.000
200.000
200.000
300.000
300.000
400.000
400.000
500.000
500.000
600.000
600.000
700.000
700.000

10µg RC P158a four times mutant, 10µg pHelper, 3,3 µg mVenus P162 were pipetted on plates: 2, 4, 6, 8, 10 , 12, 14
3,3µg RC P158a four times mutant, 3,3µg pHelper, 3,3 µg mVenus P262 were pipetted on plates: 1, 3, 5, 7, 9, 11, 13

Days	22.8	23.8	24.8	25.8	26.8	27.8	28.8	29.8	30.8
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

The results

Data were not reliable (The amount of Goi (3,3 µg instead of 10µg) was not sufficient enough)so the average YFP expression is quite low, but the remarkable note is: Beas construct (pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR) fusioned from the created Biobricks still works!

30.8 Seeding AAV293 for 10xTransfection

The motivation
We want to create ten hypothetical identical viral stocks to estimate the standard deviation.
The plan

Days	30.8	31.8	1.9	2.9	3.9	4.9	5.9	6.9
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

Transfection will be done with:

RepCap:P357 10µg =>conc.:1274,8ng/µl used amount: 7,84µl
pHelper:P356 10µg =>conc.:1068ng/µl used amount: 9,36µl
mVenus:P261 => excel sheet P263: 10µg =>conc.:979ng/µl used amount: 10,21 µl

0,5 ml of each stock become deepfreeze in.

One stock will be completely used for Transduction 9,5 ml (rest of the stock) * 0,5 ml (amount per transduction) => 19 values for estimating standard derivation.

getting valide data of 10µg samples

The motivation
we want to check the YFP expression
The plan

Days	30.8	31.8	1.9	2.9
Action	seeding HT	Transduction	-	FACS

One stock will be completely used for Transduction 9,5 ml (rest of the stock) * 0,5 ml (amount per transduction) => 19 values for estimating standard derivation.

the results

28.8 Seeding AAV293 for checking functionality of RC vector

The motivation

We want to check different R/C constructs, additionally we check if the amount of viral.!!!!!!!!!!!!!!!!! The plan

Days	28.8	29.8	30.8	31.9	1.9	2.9	3.9	4.9	5.9	6.9
Action	seeding AAV293	-	-	Transfection	-	-	Harvest+Seeding HT	Transduction	-	FACS

Transfection was done with:

RepCap:P326 => 2481 ng/µl used amount: 4µl => 10µg
RepCap:P325 => 2051 ng/µl used amount: 4,87µl => 10µg
pHelper:P356 => 684 ng/µl used amount: 414,62µl => 10µg
mVenus:P262 => 1148 ng/µl used amount: 8,7µl => 10µg

The results

Transfection will be done with:

RepCap:P357 10µg =>conc.:1274,8ng/µl used amount: 7,84µl
pHelper:P356 10µg =>conc.:1068ng/µl used amount: 9,36µl
mVenus:P261 => excel sheet P263: 10µg =>conc.:979ng/µl used amount: 10,21 µl

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4.9 checking constructs (GOIs) with/without HGH and beta globin and reassembled as control

The motivation
We want to check if these reassmbled constructs (GOIs) still work!

P269 lITR_CMV_beta-globin_mVenus_HGH_rITR = 325 ng/µl => 9,66 µl
P270 lITR_CMV_beta-globin_mVenus_HGH_rITR = 561 ng/µl => 7,26 µl
P377 lITR_CMV_mVenus_HGH_rITR = 325,04 ng/µl => 30,76 µl
P378 lITR_CMV_beta-globin_mVenus_rITR = 561 ng/µl => 17,8µl

All constructs are completed with p50 R/C =>> 26,45 µl and pHELPER 14,61µl
The plan

Days	4.9	6.9	7.9	8.9	9.9	10.9	11.9	12.9
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

6.9 checking five ganciclovir concentrations for two day incubation approach optimal medication

The motivation
we want to check the optimal ganciclovir concentration, we use the TK/GMK Stocks (10 µg from each plasmid) from 24.8 with Buffer pH 7,10 and the pH12 stock.
The plan

Days	3.9	4.9	5.9	6.9
Action	seeding HT	Transduction	-	FACS

We want to use five different ganciclovir concentrations (the concentrations are absolute to the volume in the wells):

48.5µM
97 µM
0.485 mM
0.97 mM
4.85 mM

the results

22.9 Transfection with Volkers R/C constructs

The motivation We want to check if these R/C still work!

The plan

Days	20.9	21.9	22.9	23.9	24.9	25.9	26.9	27.9	28.9
Action	seeding AAV293: 4*6wells	-	Transfection: 4 constructs	-	-	Seeding HT	Harvest + Transduction	-	FACS

The results

On the 23.9 it was shure that something went wrong with the nomenclature, so this approach is definitely not working => threwn away!

22.9 Testing of Hannas 6 VP-constructs with different compositions (10%, 25%, 50% and 75% fractions) with a VP2-knockout construct

The motivation
Test the 18 constructs for functionality on A431 and HT1080 cells.
The plan

Days	20.9	21.9	22.9	23.9	24.9	25.9	26.9	27.9	28.9
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

The results

22.9 Seeding cells for testing pTERT_mVenus in R/C P326 and P431

The motivation We want to check if the pTERT-promoter is realiable for tumor specific gene expression.

The plan

Days	22.9	23.9	24.9	25.9	26.9	27.9	28.9	29.9	30.9	1.10	2.10	3.10	4.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest	-	-	Seeding HT, A293 and A431	Transduction		FACS

The results

28.9 Seeding cells for testing loop insertions

The motivation

The plan

Days	28.9	29.9	30.9	1.10	2.10	3.10	4.10	5.10	6.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

The results

Experiment

The motivation
The plan

Days	22.9	23.9	24.9	25.9	26.9	27.9	28.9	29.9	30.9
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

The results

Citations

[1] Media:Freiburg10_Stratagene_AAV_helper_free_manual.pdf
[2] [http://www.copewithcytokines.de/cope.cgi?key=A431 Titel einfügen]
[3] Read, A. P.; Strachan, T. (1999). "Chapter 18: Cancer Genetics". Human molecular genetics 2. New York: Wiley. ISBN 0-471-33061-2.
[4] [http://www.biomol.de/dateien/infos_nr778.gif Titel einfügen]
[5] Media:Freiburg10 Titration of AAV-2 particles via a novel capsid ELISA.pdf

Team:Freiburg Bioware/NoteBook/Labjournal/Cellculture

From 2010.igem.org

Contents

Cellculture

Date of first action, title of the experiment

14.6 Seeding AAV293 for transfection

19.6 Seeding AAV293 for second transfection according to standard protocol

30.6 Examination of different Transduction methods

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

4.7 Transfection with different amounts of plasmids

14.7 Seeding AAV293 for TKGMK constructs

7.8 Seeding AAV293 for TKGMK and mVenus stocks

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

14.8 Seeding different amounts of AAV293 for transfection

17.8 transduction with viral stocks 20 µg and 40 µg

16.8 Seeding AAV293 for transfection with new R/C

20.8

19.8 Seeding AAv293 for transfection with different HBS buffers

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Checking transduction efficiency via fluorescence microscopy

25.8: Transfection of AAV293 with different amounts of cells, and two plasmid concentrations

30.8 Seeding AAV293 for 10xTransfection

getting valide data of 10µg samples

28.8 Seeding AAV293 for checking functionality of RC vector

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4.9 checking constructs (GOIs) with/without HGH and beta globin and reassembled as control

6.9 checking five ganciclovir concentrations for two day incubation approach optimal medication

22.9 Transfection with Volkers R/C constructs

22.9 Testing of Hannas 6 VP-constructs with different compositions (10%, 25%, 50% and 75% fractions) with a VP2-knockout construct

22.9 Seeding cells for testing pTERT_mVenus in R/C P326 and P431

28.9 Seeding cells for testing loop insertions

Experiment

Citations