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1 Cellculture
2 17.8 Transduction of plates with different amounts of cells
3 18.8 Transfection
4 25.8: Transfection of AAV293 with different amounts of cells, and two plasmid concentrations
- 4.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
5 Seeding AAV293 for 10xTransfection
6 getting valide data of 10µg samples
- 6.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
7 28.8 Checking functionality of RC vectors
- 7.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
8 4.9 checking constructs (GOIs) with/without HGH and beta globin and reassembled as control
- 8.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
9 6.9 checking five ganciclovir concentrations for two day incubation approach optimal medication
- 9.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
10 22.9 Transfection with Volkers R/C constructs
- 10.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
11 22.9 Testing of Hannas 6 VP-constructs with different compositions (10%, 25%, 50% and 75% fractions) with a VP2-knockout construct
- 11.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
12 22.9 Testing pTERT_mVENUS in R/C P326 and P431
- 12.1 XXXXXXXXXXXXXXXXXXXXXXXXXX
13 28.9 Testing Loop Insertions
14 Experiments
15 Citations

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: 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
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).

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.

13.8 Seeding AAV293 for Virus production

13.8 Seeding HT1080 for transduction at saturday, Seeding HEK293 for transfection

The motivation

The plan

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 results

16.8: The fluorescence was quite low (~ 5%)in nearly every well! The 40µg YFP wells were the only exception.

The conclusions
The incubation step of CaCl₂ seems to be crucial! The next transfection protocol is with a 30 min incubation on ice step! Seeding HEK293 for transfection at monday 16.8

The motivation

Which confluence of HEK293 cells is optimal for AAV production?

The plan

We seed different amounts of cells, 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 not sure

The results

Fluorescence microscopy of transduction at 14.8 transfection of plates from 13.8

Working steps:

Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C.
Pipet XXX µg of each of the three plasmid DNA solutions into an eppi. The standart protocol is: 10 µg per plasmid, experiment data showed that the amounts (3,3 µg, 6,6 µg and 10 µg per plasmid) showed similar results
Fill up to 300 µl with sterile water. Add 300µl of 0.5 M CaCl2 and mix gentlyIncubation step 45 min on ice !!!!!!
Pipet 600 µl ml of 2 × HBS into a 15-ml falcon.
Vortex the falcon (with the 2x HBS) gently!!!! while pipetting the DNA/CaCl2 solution dropwise into the falcon (use the Pipetus).
Apply the DNA/CaCl₂/HBS suspension to the plate of cells in a dropwise fashion(experimental data shows that resuspending-fashion is better for high titer), swirling gently to distribute the DNA suspension evenly in the medium.
Return the tissue culture plate to the 37°C room for 6 hours.
At the end of the incubation period, remove the medium from the plate and wash cells with warm PBS, replace it with 10 ml of fresh DMEM growth medium.This is a crucial step! The transfection-stress kills nearly all cells if theres no medium change
Return the plate to the 37°C incubator for an additional 72 hours.

Seeding HT1080

The motivation
We want to check if the amount of HT cells is crucial for Transduction (The second transduction was daone with 30.000 cells, and had a high YFP expression)

The plan
3* 6er dishes with 100.000 cells and 2* 6er dishes with 50.000 cells were seeded.
The results
FILL IN PICTURES

Seeding AAV293

The motivation
We want to create new viral stocks.
The plan
15 * 10 cm² with 2.000.000 cells on each plate were created

17.8 Transduction of plates with different amounts of cells

The plan

Platte 1 YFP: 50.000 cells per well

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

Platte 2 YFP: 50.000 cells per well

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

Platte 2 YFP: 50.000 cells per well

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

Platte 3 YFP: 100.000 cells per well

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

Platte 4 YFP: 100.000 cells per well

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

Platte 5 TKGMK: 100.000 cells per well

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

18.8 Transfection

The Motivation

Transfection with different amounts of REP/CAP and pHELPER. Additionally a The plan

XXXXXXXXXXXXXXXXXXXXXXXXXX

Transfection of AAV293 cells

Investigator: Adrian, Kerstin

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)

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)

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

21.8 Fluorescence microscopy, Transfection, AAV-Harvesting and Transduction

Transfection with different HBS-Buffers and with stratagene standard protocol

The Motivation
Transfection of AAV293 cells from 19.8 (ten 10cm² dishes with 2.000.000 cells each) with original standard protocoll!!! We want to try different 2xHBS Buffers (pH) and their influence on transfection efficiency!

The Plan

Requiered goods: 0,5 M CaCl₂ , 2x HBS-Buffer , autoclaved millipore water, falcons, eppis, sven's vortexer sterilized with EtOH
Working steps:

Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C.
Pipet 10 µg of each of the three plasmid DNA solutions into an eppi.
Fill up to 300 µl with sterile water. Add 300µl of 0.5 M CaCl2 and mix gently
Pipet 600 µl ml of 2 × HBS into a 15-ml falcon.
Vortex the falcon (with the 2x HBS) gently!!!! while pipetting the DNA/CaCl2 solution dropwise into the falcon (use the Pipetus).
Apply the DNA/CaCl₂/HBS suspension to the plate of cells in a dropwise fashion, swirling gently to distribute the DNA suspension evenly in the medium.
Return the tissue culture plate to the 37°C room for 6 hours.
At the end of the incubation period, remove the medium from the plate and wash cells with warm PBS, replace it with 10 ml of fresh DMEM growth medium.
Return the plate to the 37°C incubator for an additional 72 hours.

We try 5 different 2xHBS buffers:

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

The transfected cells were seeded at 19.8 (2.000.000 cells per 10 cm²)
Used Plasmids: Standart 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

Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C.
Pipet 10 µg of each of the three plasmid DNA solutions into an eppi.
Add 1000µl of 0.3 M CaCl2 and mix gently
Pipet 1000 µl ml of 2 × HBS into a 15-ml falcon.
Pipett the DNA/CaCl2 solution dropwise into the falcon (use the Pipetus).
Apply the DNA/CaCl₂/HBS suspension to the plate of cells in a dropwise fashion, swirling gently to distribute the DNA suspension evenly in the medium.
Return the tissue culture plate to the 37°C room for 6 hours.
At the end of the incubation period, remove the medium from the plate and wash cells with warm PBS, replace it with 10 ml of fresh DMEM growth medium.
Return the plate to the 37°C incubator for an additional 72 hours.

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

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

XXXXXXXXXXXXXXXXXXXXXXXXXX

Harvesting viral stock from 18.8.2010 and following transduction

the motivation

We want to check the efficiency of Lipo-transfection and standard transfection with different ratios of REPCAP/pHelper, additionally if the modified RC (p158a and p158b) is still functionally.

The plan

Harvesting the cells with the Stratagene-standard protocoll.

The results

13 virals stocks were created (in 2.000.000 cells per 10cm² dish):

Lipo-Transfection 1 (2µl pHelper + 0,8µl RC + 6,2 µl YFP)
Lipo-Transfection 2 (2µl pHelper + 0,8µl RC + 6,2 µl YFP)
3,3 µg of RC, pHelper and mVenus
10 µg RC, 10 µg pHelper, 3,3 µg GOI
10µg RC P158a four times mutant, 10µg pHelper, 3,3 µg mVenus
10µg RC P158b four times mutant, 10µg pHelper, 3,3 µg mVenus
3,3µg RC P158a four times mutant, 3,3µg pHelper, 3,3 µg mVenus
3,3µg RC P158b four times mutant, 3,3µg pHelper, 3,3 µg mVenus
10µg RC, 10µg pHelper, 3,3µg GOI (broken arrow)
10µg RC, 10µg pHelper, 10µg GOI (broken arrow)
10µg RC, 10µg pHelper, 20µg GOI (broken arrow)
10µg RC, 10µg pHelper, 40µg GOI (broken arrow)
3,3 µg RC, 3,3 µg pHelper, 3,3 µg GOI
negative control
negative control

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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

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

The motivation

We want to define the final AAV293-concentration 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!

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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

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28.8 Checking functionality of RC vectors

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

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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

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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!

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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

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22.9 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

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28.9 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

Experiments

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] 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] Titel einfügen
[5] Media:Freiburg10 Titration of AAV-2 particles via a novel capsid ELISA.pdf

@@ Line 594: / Line 594: @@
 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.
-===<p style="font-size:17px; background-color:#00dd77;">12.8 Seeding AAV293 for Virus production </p>===
+===<p style="font-size:17px; background-color:#00dd77;">13.8 Seeding AAV293 for Virus production </p>===
-<br />
-<b>The motivation</b><br />
-In first place 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).<br />
-<b>The plan</b><br />
-See transduction protocoll for further information, the final centrifugation step was done with 2000 g 5 min.<br />
-<b>The results</b><br />
-Ten viral stocks with 3 ml each were prepared.
-<br />
-{| border="1"
-| Plate  || align="right"  |pAAV_RC/µg|| align="right" |pHelper/µg|| align="right" |GOI/µg
-|-
-| 1||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | no GOI
-|-
-| 2||  align="right" |6,6 ||  align="right" | 3,3||  align="right" | no GOI
-|-
-| 3||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 TK_GMK clone1
-|-
-| 4||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 TK_GMK clone1
-|-
-| 5||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 TK_GMK clone2
-|-
-| 6||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 YFP
-|-
-| 7||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 10 YFP
-|-
-| 8||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 20 YFP
-|-
-| 9||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 40 YFP
-|-
-| 10|| align="right" |3,3 ||  align="right" | 3,3||  align="right" | 60 YFP
-|-
-|}
-<br />
-<b>The nomenclature of the plasmids is wrong, the date is 12.8. not 13.8</b>
 ===<p style="font-size:17px; background-color:#00dd77;">13.8 Seeding HT1080 for transduction at saturday, Seeding HEK293 for transfection</p>===

Team:Freiburg Bioware/NoteBook/Labjournal/Cellculture

From 2010.igem.org

Revision as of 15:16, 9 October 2010

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

13.8 Seeding AAV293 for Virus production

13.8 Seeding HT1080 for transduction at saturday, Seeding HEK293 for transfection

Fluorescence microscopy of transduction at 14.8 transfection of plates from 13.8

Seeding HT1080

Seeding AAV293

17.8 Transduction of plates with different amounts of cells

18.8 Transfection

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Transfection of AAV293 cells

20.8

21.8 Fluorescence microscopy, Transfection, AAV-Harvesting and Transduction

Transfection with different HBS-Buffers and with stratagene standard protocol

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Harvesting viral stock from 18.8.2010 and following transduction

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Checking transduction efficiency via fluorescence microscopy

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

XXXXXXXXXXXXXXXXXXXXXXXXXX

Seeding AAV293 for 10xTransfection

getting valide data of 10µg samples

XXXXXXXXXXXXXXXXXXXXXXXXXX

28.8 Checking functionality of RC vectors

XXXXXXXXXXXXXXXXXXXXXXXXXX

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

XXXXXXXXXXXXXXXXXXXXXXXXXX

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

XXXXXXXXXXXXXXXXXXXXXXXXXX

22.9 Transfection with Volkers R/C constructs

XXXXXXXXXXXXXXXXXXXXXXXXXX

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

XXXXXXXXXXXXXXXXXXXXXXXXXX

22.9 Testing pTERT_mVENUS in R/C P326 and P431

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28.9 Testing Loop Insertions

Experiments

Citations