From 2010.igem.org

(Difference between revisions)

Latest revision as of 21:38, 27 October 2010

NoteBook Navigator

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

Days	14.6	15.6	16.6	17.6	18.6	19.6	20.6	21.6
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		Fluorescence microscopy

AAV 293 and HT1080 Cells have been splitted and plated out on 10 cm dishes for transfektion. After harvesting the cells according to the standard protocol the cell-pellets were resuspendiated in 15ml of DYT medium. 2,5µl of this cell suspension have been mixed with 47.5µl of trypan blue and the cells were counted by using the Neubauer-Meteringchamber.

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

1ml and 1,5 ml of the AAV 293 cells have been seeded on four dishes with 250µl of the cell suspension. 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 2ml 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 were 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 and obtain a vsibly higher GOI (mVenus) expression.
The plan

Days	19.6	20.6	20.6	21.6	22.6	23.6	24.6	25.6
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		Fluorescence microscopy

Six transfections were performed according to the standart protocol:
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 check whether the amount of our produced AAV2s has an effect on the transduction rate. Instead of 500µl, 1000µl of the AAV-stock were pipetted into each well.

The plan

Days	30.6	1.7	2.7	3.7	4.7	5.7	6.7	7.7
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		Fluorescence microscopy

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 rough resuspending
1000 µl of Viral Stock DROPPED	1000 µl of Viral Stock gently resuspended	no Transduction

The results
All cells died probably because we forgot to wash the cells with PBS before adding fresh medium. It was not possible to take pictures because the dead cells were all clumped. 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. Unfortunately the flow cytometry (FACS) is not available at the moment so the results can only be checked via fuorescence microscopy.

The plan

Days	3.7	4.7	5.7	6.7	7.7	8.7	9.7	10.7
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		Fluorescence microscopy

Transfection with P41 (pAAV_iGEM-MCS_mVenus) 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 GOI-plasmid (mVenus)
we have 10µg and 20µg from standart protocol => standard virus
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 and transduction efficiency can be improved with higher amounts of DNA.

The plan

Days	4.7	5.7	6.7	7.7	8.7	9.7	10.7	11.7
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

The Transfection was performed with 10, 18 and 24 µg per plasmid (mVenus, pHelper and R/C).
The virus harvest was performed according to standard protocol:

3 Plates got transduced

1. Plate I(A is up) completely 10 µg

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

2. Plate II(A is up)completely 18 µg

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

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

300µl resuspended	300µl resuspended	450µl resuspended
600µl resuspended	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%

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

Days	14.7	15.7	16.7	17.7	18.7	19.7	20.7	21.7
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

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 (exception: see following experiment, 7. august) because later on we noticed that the TKGMK plasmids were not in line with the RFC.

7.8 Seeding AAV293 for TKGMK and mVenus stocks

The motivation After proofing the successfull transduction of tumor cells with the reportergen mVenus the next step is to kill the cells with a prodrug approach. The prodrug of our choice is the guanosine anolog ganciclovir, once activated ganciclovir gets integrated into the growing dna chain which results in termination of dna synthesis and cell death. We transduce the tumor cell line HT1080 with viral particles packed with the thymidine kinase guanosine mono phosphate kinase fusion protein (TKGMK) as gene of interest and the incubation with ganciclovir should kill the cells.

The plan

Days	7.8	8.8	9.8	10.8	11.8	12.8	13.8	14.8
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS and microscopy

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 (P54)
4	3,3	3,3	3,3 TK_GMK clone1 (P54)
5	3,3	3,3	3,3 TK_GMK clone2 (P55)
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

The transduction was performed according to standard protocol.

Plate 1:

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

Plate 2:

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

Plate 3:

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

Plate 4:

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

Plate 5:

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

Plate 6:

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

Plate 7:

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

Plate 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.

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

Days	14.8	15.8	16.8	17.8	18.8	19.8	20.8	21.8
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

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

Plate	amount of seeded AAV-293 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 standard CMV_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)

The results
The FACS was still not available but we could easily detect a significant higher YFP expression (70-80%) at the following positions:

Plate 1: A2 and B2, A3 and B3
Plate 2: A2 and B2, A3 and B3

Therefore we conclude that we have to seed between 100000 and 500000 cells for optimal virus production precedure.

17.8 transduction with viral stocks 20 µg and 40 µg GOI-plasmid

The Motivation
We want to FACS our stocks to get valid data for further investigation of optimal virus assembly. Therefore created stocks with 20 and 40 µg CMV_mVenus.
The Plan

Days	17.8	18.8	19.8	20.8	21.8	22.8	23.8	24.8
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

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)

The Results
Although we were told that the FACS would be available in time it wasn't. So the cells could unfortunately not be checked for their YFP expression =(.

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 AAV-293 cells which were thawed.

The plan

Days	16.8	17.8	18.8	19.8	20.8	21.8	22.8	23.8
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

2x10⁶ cells were seeded
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 => named Lipo
2. plate: Lipo-Transfection => named Lipo
3. plate: 3,3 µg (RC), 3,3 µg (pHelper), 3,3 µg (YFP) => was not used for transduction
4. plate: 10 µg (RC), 10 µg (pHelper), 3,3 µg (YFP) => named CaCl₂
5. plate: 10 µg (RC mutant P158a), 10 µg (pHelper), 3,3 µg (YFP)=> CaCl₂ 10 µg RC mutant P158a
6. plate: 10 µg (RC mutant P158b), 10 µg (pHelper), 3,3 µg (YFP)=> CaCl₂ 10 µg RC mutant P158b
7. plate: 3,3 µg (RC mutant P158a), 3,3 µg (pHelper), 3,3 µg (YFP)=> CaCl₂ 3,3 µg RC mutant P158a
8. plate: 3,3 µg (RC mutant P158b), 3,3 µg (pHelper), 3,3 µg (YFP)=> CaCl₂ 3,3 µg RC mutant P158b
9. plate: 10 µg (RC), 10 µg (pHelper), 3,3 µg (eGFP)=> CaCl₂ eGFP 3,3 µg
10. plate: 10 µg (RC), 10 µg (pHelper), 10 µg (eGFP)=> CaCl₂ eGFP 10 µg
11. plate: 10 µg (RC), 10 µg (pHelper), 20 µg (eGFP)=> CaCl₂ eGFP 20 µg
12. plate: 10 µg (RC), 10 µg (pHelper), 40 µg (eGFP)=> CaCl₂ eGFP 40 µg

The results

As you can see the eGFP constructs worked very well. And even the transduction efficiency of the standard R/C is ok, the conclusion is that in previous transfections the AAV293 cells were to confluent so their transfection capability. The confluence of the cells is crucial! It also seems that our used amounts of plasmids had no decisive effect on the YFP expression because of the obviously variating results. A final conclusion is still due.

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

Days	19.8	20.8	21.8	22.8	23.8	24.8	25.8	26.8
Action	seeding AAV293	-	Transfection	-	Seeding HT	Harvest + Transduction		FACS

We try 5 different 2xHBS buffers:

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

Used Plasmids: 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

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)=7,112
(4)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,12
(5)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,14

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 7.112 performed best, in future experiments this buffer will be used.

22.8: 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

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 following amount of cells were seeded in 10 cm²:

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

The results

There is only one value for each approach but nevertheless the average YFP expression should be higher. We really dont know the reason for the quite low YFP expression. There are also very good news: the reassembled construct (pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR) created from the single Biobricks works as "well" as the AAV2 with CMV_mVenus!

30.8 Seeding AAV293 for Transfection of 10 cm² plates

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 be stored at -80°C.

9,5 ml of each stock will be used for Transduction.

Result:

Standard deviation: about +/- 4,98% YFP expression. All future AAV2 stocks will be pooled if produced with this same plasmids and amounts of plasmids. Unfortunately the YFP expression is still located at around 50%. We would like to have a YFP expression of at least 70% because as soon as we start to test our not yet ready modified AAV2 we suggest the YFP expression will obviously decrease.

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

The motivation
We want to check the functionality of these genes of interest

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

P269 pSB1C3_lITR_CMV_beta-globin_mVenus_HGH_rITR = 325 ng/µl => 9,66 µl
P270 pSB1C3_lITR_CMV_beta-globin_mVenus_HGH_rITR = 561 ng/µl => 7,26 µl
P377 pSB1C3_lITR_CMV_mVenus_HGH_rITR = 325,04 ng/µl => 30,76 µl
P378 pSB1C3_lITR_CMV_beta-globin_mVenus_rITR = 561 ng/µl => 17,8µl

Additionally P50: pAAV_RC R/C (26,45 µl) and pHELPER (14,61µl) pipetted to each approach.

The results

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

As expected the highest concentration of ganciclovir worked the best but it is remarkable that there is no significant difference between the first three ganciclovir amounts. The most remarkable note is, that in future experiments the incubation time has to be extended to 3 or 4 days.

15.9 Seeding AAV293 for testing our Affibody and other VP-mVenus constructs

The motivation
The pCerulean_VP1up_mVenus_Vp2/3 Construct will be tested in three different compositions.

The plan
We want to test these constructs:

pCerulean_VP1up_NLS_Affibody_VP2/3
pCerulean_VP1up_His_VP2/3
pCerulean_VP1up_mVenus_Vp2/3

The His-tag is for purify our constructs and in this case we want to test, if the particles are still infectious. Construct with the affibody will be tested for functionality and even for their better affinity to our EGFR overexpressing cell line A431.

The results
Interpretation and discussion of our results.

19.9 Seeding AAV293 for checking two different Rep/Cap Constructs

The motivation
During the project 22 silent nucleotide exchange mutations were introduced into the capsid coding construct to make it compatible with the RFC standards and to have single cutting restriction enzymes flanking the 453 and the 587 loop sequence. Two point mutations had to be dismissed, because either a first test transduction showed that the construct was not working anymore or the insertion of the synthesized gene posed serious problems, because the restriction enzyme did not work.
The plan
Transfection and transduction was performed according to standard protocol.

The results

Finally, we have a construct that is shown to produce infectious particles comparable to the current AAV systems and carries 20 point mutations. Now we can announce that the Adeno-associated Virus is compatible to the RFC standard and the idea to replace the loop sequences via ViralBricks works!

22.9 Seeding AAV293 for transfection with different mVenus constructs for checking specific promoter activity

The motivation
We want to check if the CMV and pTERT promoters work equaly in the AAV293, A431 and HT1080 cells. So we transfect them via standard transfection CaCl₂. It is obvious that the individual cell lines are not equaliy competent to transfection (the AAV293 are optimised for transfection). It seems that this promoter is not that active in AAV293 cells.
The plan
We transfected the three cell lines A431, AAV293 and HT1080 with following three constructs:

CMV_mVenus
CMV_eGFP
pTERT_mVenus

Remember, that mVenus and eGFP have the exact same excitation wavelenght. We took the pictures with the same parameters, but saved the data in the wrong formate (.tif instead of .zvi), so we werent able to change the YFP expression from green to yellow.

Days	22.9	23.9	24.10	25.10	26.10
Action	seeding AAV293	-	Transfection	fluorescence microscopy	fluorescence microscopy

Transfection of different cell types with different promoter_reportergenes
AAV293 CMV_eGFP 16h post transfection	AAV293 CMV_mVenus 16h post transfection
AAV293 pTERT_mVenus 16h post transfection	AAV293 CMV_eGFP 48h post transfection
HT1080 CMV_mVenus 48h post transfection	A431 CMV_mVenus 48h post transfection

The results
As excepted, the expression of the CMV_mVenus and CMV_eGFP was very high even at 16h post transfection. The pTERT_mVenus construct worked aswell, but significantly weaker! Problematic is the fact, that there was no expression in HT1080 and A431 detectable. Due the fact that these cell lines are not optimized for CaCl₂ transfection. We can't evaluate if the bad transfection efficiency or the low promoter activity of pTERT are responsible for the results.

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 following 18 constructs for functionality on A431 and HT1080 cells. Each of these constructs getting transfected in different compositions 25%, 50%, 75% and 90% with VP2_knockout 75%. In this way we are able to create viral particles with this ratios of VP proteins. We hope that the created particles are still infectious.

pCerulean_CFP_MiddleLinker_VP2/3insCap P528
pCerulean_Zegfr:1907_ShortlLinker_VP2/3insCap P527
pCerulean_6xHis_Middlelinker_VP2/3insCap P534
pCerulean_Zegfr:1907_Middlelinker_VP2/3insCap P535
pCerulean_Zegfr:1907_SEG_VP2/3_Capins 10:90 P502
pCerulean_Zegfr:1907_LongLinker_VP2/3_CapIns P503

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

Obviously the constructs still work!

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, we use two different kinds of R/C.

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

29.9 Checking location of VP proteins via fluorescence microscopy and checking fractions of centrifugation steps via fluorescence spectrometer

The motivation: We want to figure out if it is possible to purify our viral stocks via centrifugation with 10.000 g and/or 20.000 g. In theory/ according to the literature the viral particles should be in the cells and attached to the HSPGs at the cellular surfaces.
The plan: Two different viral stocks were prepared:

Stock 1 with VP2-mVenus-fusion-Capsid loaded with TKGMK (4 wells from a 6 well plate)

R/C 1: 50% pCerulean_VP1up_NLS_mVenus_Vp2/3 P501
R/C 2: 50% Rep/Cap2: P449 pAAV_RC_4fachmut_VP1-ko:
GOI: TKGMK: P82.b
pHelper

Stock 2 "standard" virus (4 wells from a 6 well plate)

R/C 1 P486
GOI: TKGMK: P82.b
pHelper

These two stocks got harvested according to standard protocol (scratching cells, transfer them in to 15 ml falcons, resuling 12ml solution total). After centrifugation (10 min 200g), the supernatant got transferred into two new 15 ml falcons and the pellets were resuspended with 10 ml DMEM Medium. The Stocks got freezed thawed two times so finally there were:

DMEM as negative control
resuspended Pellet with mVenus-fusioned viral capsids
resuspended Pellet with "standard virus"
supernatant with mVenus-fusioned viral capsids
supernatant with "standard virus"

The fluorescence of each stock was measured via spectrometer. After that, stocks 2 and 4 were centrifugated 10min with 10.000 g followed by an other fluorescence measuring. Finally the stocks got spin down with 20.000 g for 10 min
The results:

Transfection with VP-mVenus-fusion constructs
Picture two days post transfection.

Freiburg10 mVenus VP fusion fluorescence centrifugation experiment graph.jpg

Freiburg10 mVenus VP fusion fluorescence centrifugation experiment 526nm.jpg

The conclusions: The highest amount of fluorescence was measured in the supernatant, the individual centrifugation steps had no decrease in fluorescence as consequence!

Keep in mind, without further investigation it is not valid to say that we can purify our stocks via 10-20.000 g centrifugation steps, because we dont know if the viral capsids are still intact! The FACS analysis are not sufficient enough to make a decision, because our last samples had YFP as GOI (=> so efficient cell sorting was not possible). We need to do qPCRs to make a valid evidence.

30.9 Production of new Standard mVenus Vector

The motivation
The AAV2 with 4 point mutations, inserted rep and cap and remutated KpnI restriction has to be checked for its functionality in order to compare it with the wild type AAV2. Hopefully there will be no significant difference.
The plan
After the "delay" 200.000 A431 and HT1080 cells were seeded and transduced on the following day with 0,5 ml of this AAV2 mutant.

Days	30.9	1.10	2.10	3.10	4.10	5.10	6.10	7.10	8.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest	delayed because of cell death	delayed because of cell death

The results
The stocks were harvested successfully

30.9 Seeding AAV293 for production of standard TKGMK vector

The motivation
We want to create a big amount of viral particles with TKGMK as gene of interest for using it as an standard vector for the MTT-assay.
The plan

Days	30.9	1.10	2.10	3.10	4.10	5.10	6.10	7.10	8.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest	delayed because of cell death	delayed because of cell death

The results
The viral stocks were harvested efficiently and are ready to use.

30.9 Seeding AAV293 for production of vectors with mVenus missing beta globin

The motivation
Beta globin is said to improve (stratagene) the expression so we expect a worse mVenus (reportergene) expression compared to the standard AAVs. Therefore the seeded A431 and HT1080 cells were transduced with a AAV2 mutant missing beta-globin.
The plan
Ater the "delay" 200.000 HT1080 and A431 cells were seeded and transduced with 0,5 ml AAV2 mutant on the following day.

Days	30.9	1.10	2.10	3.10	4.10	5.10	6.10	7.10	8.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest	delayed because of cell death	delayed because of cell death

The results
Interpretation and discussion of our results.

30.9 Seeding AAV293 for production of vectors with mVenus missing HGH

The motivation
HGH is responsible for the mRNA polyadenylation of the virus so we expect expect a worse mVenus (reportergene) expression compared to the standard AAVs. Therefore the seeded A431 and HT1080 cells were transduced with a AAV2 mutant missing HGH.
The plan
After the "delay" 200.000 A431 and HT1080 cells were seeded and transduced on the following day with 0,5 ml AAV2 mutant missing HGH.

Days	30.9	1.10	2.10	3.10	4.10	5.10	6.10	7.10	8.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest	delayed because of cell death	delayed because of cell death

The results
Interpretation and discussion of our results.

30.9 Seeding AAV293 for testing RC with HSPG knockout for functionality

The motivation
The primary AAV2 receptor (the HSPG receptor) was knocked out so we expect a worse tranduction rate (about 0,3 to 0,4 of the standard transduction rate, assuming that there are HSPGs on the HT1080 and A431 cell surface). The worse the better.
The plan
After the "delay" 200.000 A431 and HT1080 cells were seeded and transduced on the following day with 0,5 ml AAV2 mutant with a HSPG-receptor knockout.

Days	30.9	1.10	2.10	3.10	4.10	5.10	6.10	7.10	8.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest	delayed because of cell death	delayed because of cell death

The results

Freiburg10 HT1080 missing HSPG binding motif.png

Freiburg10 A431 missing HSPG binding motif.png

There is a significant difference in transduction efficiency. The actual reduction is even higher than expected, according to literature it should be around 60%, we reached 80%. We measured via qPCR the amount of viral particles for each stock, and they are nearly the same the HSPG Knockout has 2*10⁷particles per ml , the stock with HSPG-binding motif has 4.5*10⁶ particles per ml.

30.9 Seeding AAV293 for testing RC WITHOUT HSPG knockout for functionality

The motivation
The produced vector has the intact HSPG-binding motif, so we can compare it with the vector produced in: "Seeding AAV293 for testing RC with HSPG knockout for functionality"
The plan
Transfection and transduction were performed according to standard protocol.

Days	30.9	1.10	2.10	3.10	4.10	5.10	6.10	7.10	8.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest	delayed because of cell death	delayed because of cell death

The results
See 30.9 Seeding AAV293 for testing RC with HSPG knockout for functionality

12.10 Seeding AAV293 cells for transfection with VP-BAP

The motivation
We want to create a viral stock for purification and biotinylation, the ratio of the Rep/Cap plasmids is 1:1.
The plan
Transfection was performed according to standard protocol:

Gene of interest: mVenus
R/C: pSB1C3-001_pCMV_[AAV]VP123_587_BAP in cotransfection with pSB1C3_001_RC_IRCK_P5tataless clone 1

Days	12.10	13.10	14.10	15.10	16.10	17.10	18.10	19.10	20.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

The results

12.10 Seeding AAV293 cells for transfection with mVenus as gene of interest and pCerulean_VP1up_NLS_mVenus_VP2/3_HSPG-KO

The motivation
We want to create a viral stock for purification and biotinylation, the ratio of the Rep/Cap plasmids is 1:1.
The plan
Transfection was performed according to standard protocol:

Gene of interest: mVenus
R/C: pCerulean_VP1up_NLS_mVenus_VP2/3_HSPG-KO in cotransfection with pSB1C3_001_RC_IRCK_VP1-ko_HSPG-ko_P5tataless cl1

Days	12.10	13.10	14.10	15.10	16.10	17.10	18.10	19.10	20.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

The results

12.10 Seeding AAV293 cells for transfection with mVenus as GOI and CFP_Middlelinker_VP2/3_HSPG-KO

The motivation
This viral stock is for live imaging and probably for western blotting, the ratio of the Rep/Cap plasmids is 1:1.
The plan

Days	18.10	19.10	20.10	21.10	22.10	23.10	24.10	25.10	26.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

Transfection was performed according to standard protocol:

Gene of interest: mVenus
R/C: CFP_Middlelinker_VP2/3_HSPG-KO in cotransfection with pSB1C3_001_RC_IRCK_P5tataless clone 1

The results

17.10 Production of AAV2 with 587-His

Investigator Patrick

The motivation
We wanted to produce AAV2 which could be purified via affinity purification. Therefore 10 x 10cm^2 were seeded with AAV-293. Five of these plates contained serum-free medium and cells accustomized to this.
Used plasmids:

pHelper
pSB1C3_leftITR_CMV_betaglobin_mvenus_hGH_rightITR (P541)
50% pSB1C3_001_RC_IRCK_P5tataless (P712)
50% pSB1C3-001_pCMV_[AAV2]VP123_587_HIS (P638)

The plan
In this section, the design of the experiment is explained.

Days	17.10	19.10	20.10	21.10	22.10	23.10	24.10	25.10	26.10
Action	seeding AAV293	Transfection	-	Seeding HT	AAV2 harvest	Transduction		FACS

The results
Interpretation and discussion of our results.

18.10 Seeding AAV293 cells for transfection Superconstructs

The motivation
We want to create a viral stock for purification and biotinylation, the ratio of the Rep/Cap plasmids is 1:1.
The plan

Days	18.10	19.10	20.10	21.10	22.10	23.10	24.10	25.10	26.10
Action	seeding AAV293	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

Transfection was performed according to standard protocol:

Gene of interest: CMV_mVenus

pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis in ratio 1:1 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1
pSB1C3-001_pCMV_[AAV2]VP123_453_Z34C in ratio 1:1 pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10
pSB1C3_001_CMV_VP123_587-KO_Z34C in ratio 1:1 pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10
pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer in ratio 1:1 pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10

Gene of interest: pTERT_TKGMK

pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis in ratio 1:1 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1

Gene of interest: CMV_TKGMK

pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis in ratio 1:1 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1

The results

18.10 Seeding AAV293 cells for transfection with Cytosindeaminase

The motivation

We want to create viral stocks with CMV_Cytosindeaminase and pTERT_Cytosindeaminase as gene of interests, to check qualitatively the bystander effect.

The plan

Days	18.10	19.10	20.10	21.10	22.10	23.10	24.10	25.10	26.10	27.10
Action	seeding AAV293	-	-	Transfection	-	-	Seeding HT	Harvest + Transduction	-	FACS

Transfection, harvest and transduction were performed according to standard protocol:

Gene of interest: CMV_Cytosindeaminase

P712

Gene of interest: pTERT_Cytosindeaminase

P712

The results

22.10.2010 Transduction with old mVenus Stocks for taking pictures

The motivation We transduced HT1080 with our stocks packaged with CMV_mVenus.
The plan

Days	22.9	23.9	24.9	25.9
Action	seeding HT1080	Transduction	-	Fluorescence microscopy

The results

Transduction with vectors pSB1C3_001_RC_IRCK_P5tataless as R/C and CMV_mVenus

23.10 Testing the bystander effect of the cytosine deaminase

The motivation

The activated form of 5-fluorocytosine has the ability to diffuse through the plasma membrane. In this way cells get killed which were not transduced by the viral particles. We use an old yfp stock t determine if the transgene expression is on and to harvest the cells which are transduced with cytosine deaminase loaded AAV2.

The plan

Days	23.10	24.10	25.10	26.10	27.10	28.10
Action	Transduce HT1080 and seed HT1080	Transfer the transduced HT1080 onto the non transduced HT1080	-	-	-	Measure living cells via Neubauer cellchamber

Gene of interest: CMV_Cytosindeaminase

P712

Gene of interest: CMV_mVenus

P468

All dishes had this order:

	1	2	3
A	Medium with prodrug	Medium with prodrug	Medium with prodrug
B	Medium	Medium	Medium

The concentration of the prodrug 5-fluorocytosine is 54 mmolar

Cells were seeded following this scheme:
plate 1

	1	2	3
A	1*10⁵ HT1080 transduced with CMV_Cytosindeaminase	1*10⁵ HT1080 transduced with CMV_Cytosindeaminase	1*10⁵ HT1080 transduced with CMV_Cytosindeaminase
B	1*10⁵ HT1080 transduced with CMV_Cytosindeaminase	1*10⁵ HT1080 transduced with CMV_Cytosindeaminase	1*10⁵ HT1080 transduced with CMV_Cytosindeaminase

plate 2

	1	2	3
A	2*10⁵ HT1080 non transduced	2*10⁵ HT1080 non transduced	2*10⁵ HT1080 non transduced
B	2*10⁵ HT1080 non transduced	2*10⁵ HT1080 non transduced	2*10⁵ HT1080 non transduced

The next plates were seeded with non transduced cells and 1*10⁵ of the cytosin deaminase transduced cells
plate 3

	1	2	3
A	2*10⁵ HT1080	2*10⁵ HT1080	2*10⁵ HT1080
B	2*10⁵ HT1080	2*10⁵ HT1080	2*10⁵ HT1080

The results

25.10.2010 Transduction with several constructs for Flow Cytometry

The motivation We want to quantify via flow cytometry the Affibody superconstruct, Darpin HSPG-KO Construct and standard R/C with HSPG-KO.
The plan

Days	25	26	27
Action	Transduction	-	FACS

The results

BLANK EXPERIMENT

The motivation
The plan

Days	-	-	-	-	-	-	-	-	-
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

@@ Line 1: / Line 1: @@
 {{:Team:Freiburg_Bioware/css}}
-{{:Team:Freiburg_Bioware/SubNoteBook}}
 {{:Team:Freiburg_Bioware/Head}}
+{{:Team:Freiburg_Bioware/menu_notebook}}
 <!-- Freiburg_bioware -->
@@ Line 20: / Line 20: @@
 <li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
 <li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 145 )</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 146 - 155 )</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October3">October part 3 (labday 156 - 166 )</a></li>
 <li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture </a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
 </ul>
 </div>
@@ Line 55: / Line 54: @@
 <br />
 <b>The plan</b><br />
-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.
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-After harvesting by following the standard protocol the pallet have been resuspendiated in 15ml of DTT medium.
+<tr>
-,5µl of this cell suspension have been mixed with 47.5µl of trypan blue.
+<th width="100">Days</th>
+<th width="100">14.6</th>
+<th width="100">15.6</th>
+<th width="100">16.6</th>
+<th width="100">17.6</th>
+<th width="100">18.6</th>
+<th width="100">19.6</th>
+<th width="100">20.6</th>
+<th width="100">21.6</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>Fluorescence microscopy</td>
+</tr>
+</table>
+AAV 293 and HT1080 Cells have been splitted and plated out on 10 cm dishes for transfektion. After harvesting the cells according to the standard protocol the cell-pellets were resuspendiated in 15ml of DYT medium.
+,5µl of this cell suspension have been mixed with 47.5µl of trypan blue and the cells were counted by using the Neubauer-Meteringchamber.
 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
+ml and 1,5 ml of the AAV 293 cells have been seeded on four dishes with 250µl of the cell suspension.
-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.
-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.
+We also seated two flasks one for each cell line. Therefor we used 1ml of the HT1080 cell suspension and 2ml of the the AAV293 cells.
 <br />
 )
@@ Line 90: / Line 111: @@
 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.
 <br />
-The viral stocks are harvested according to standard protocol.
+The viral stocks were harvested according to standard protocol.
 <br>
 <b>The results</b><br />
@@ Line 106: / Line 127: @@
 <br />
 <b>The motivation</b><br />
-We want to repeat the last transfection to exclude faults in practical
+We want to repeat the last transfection and obtain a vsibly higher GOI (mVenus) expression.
 <br />
 <b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">19.6</th>
+<th width="100">20.6</th>
+<th width="100">20.6</th>
+<th width="100">21.6</th>
+<th width="100">22.6</th>
+<th width="100">23.6</th>
+<th width="100">24.6</th>
+<th width="100">25.6</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>Fluorescence microscopy</td>
+</tr>
+</table>
 Six transfections were performed according to the standart protocol:<br>
 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).
@@ Line 133: / Line 178: @@
 The Transduction efficency is still quite low! We have to optimize the stratagene protocol!
-===<p style="font-size:17px; background-color:#00dd77;">30.6 Examination of different Transduction methods </p>===
+===<p style="font-size:17px; background-color:#00dd77;">30.6 Examination of different Transduction methods</p>===
 <br />
 <b>The motivation</b><br />
-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!
+We want to check whether the amount of our produced AAV2s has an effect on the transduction rate.
+Instead of 500µl, 1000µl of the AAV-stock were pipetted into each well.
 <br /><br />
 <b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">30.6</th>
+<th width="100">1.7</th>
+<th width="100">2.7</th>
+<th width="100">3.7</th>
+<th width="100">4.7</th>
+<th width="100">5.7</th>
+<th width="100">6.7</th>
+<th width="100">7.7</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>Fluorescence microscopy</td>
+</tr>
+</table>
 . Plate I(A is up)
@@ Line 151: / Line 221: @@
 {| border="1"
-| 1000 µl of Viral Stock  DROPPED || align="right" |1000 µl of Viral Stock gently resuspended|| align="right" |1500 µl of Viral Stock hard resuspending
+| 1000 µl of Viral Stock  DROPPED || align="right" |1000 µl of Viral Stock gently resuspended|| align="right" |1500 µl of Viral Stock rough resuspending
 |-
 | 1000 µl of Viral Stock  DROPPED ||  align="right" | 1000 µl of Viral Stock gently resuspended||  align="right" | no Transduction
 |-
 |}
 <br>
-. Plate II (A is up)
-{| border="1"
-|  about 3x10^5 cells  || align="right" |only medium|| align="right" |only medium
-|-
-| about 3x10^5 cells  ||  align="right" |only medium||  align="right" |only medium
-|-
-|}
-<br>
-Transduction:
-<br>
-{| border="1"
-|  1500 µl of Viral Stock gently resuspended  || align="right" |no Transduction|| align="right" |no Transduction
-|-
-|  1500 µl of Viral Stock gently resuspended  ||  align="right" |no Transduction||  align="right" |no Transduction
-|-
-|}
-<br />
 <b>The results</b><br />
-All cells are dead. It was not possible to take pictures because the dead cells were all clumped up.
+All cells died probably because we forgot to wash the cells with PBS before adding fresh medium. It was not possible to take pictures because the dead cells were all clumped.
 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.
 <br>
@@ Line 188: / Line 240: @@
 <br />
 <b>The motivation</b><br />
-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.
+We want to check if the amount of plasmids is the reason for the low transduction efficiency. Unfortunately the flow cytometry (FACS) is not available at the moment so the results can only be checked via fuorescence microscopy.
 <br /><br />
 <b>The plan</b><br />
-Transfection with P41 was performed according standart protocol.
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">3.7</th>
+<th width="100">4.7</th>
+<th width="100">5.7</th>
+<th width="100">6.7</th>
+<th width="100">7.7</th>
+<th width="100">8.7</th>
+<th width="100">9.7</th>
+<th width="100">10.7</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>Fluorescence microscopy</td>
+</tr>
+</table>
+Transfection with P41 (pAAV_iGEM-MCS_mVenus) 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).<br />
 We want to investigate which of the following approaches yields a better transduction efficiency.
@@ Line 206: / Line 282: @@
 * 3 cycles of freezing and thawing
 <br />
-* used plasmids: 10µg, 20µg YFP
+* used plasmids: 10µg, 20µg GOI-plasmid (mVenus)
-* we have 10µg and 20µg from standart protocol => SV
+* we have 10µg and 20µg from standart protocol => standard virus
 * we have 10µg and 20µg from standart protocol pellet => Pellet
 * we have 10µg and 20µg from standart protocol suspension => Super
@@ Line 253: / Line 329: @@
 <br />
 <b>The motivation</b><br />
-We want to check if the mVenus expression/ transduction efficiency can be extended with higher amounts of DNA.
+We want to check if the mVenus expression and transduction efficiency can be improved with higher amounts of DNA.
 <br /><br />
 <b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">4.7</th>
+<th width="100">5.7</th>
+<th width="100">6.7</th>
+<th width="100">7.7</th>
+<th width="100">8.7</th>
+<th width="100">9.7</th>
+<th width="100">10.7</th>
+<th width="100">11.7</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>FACS</td>
+</tr>
+</table>
 The Transfection was performed with 10, 18 and 24 µg per plasmid (mVenus, pHelper and R/C).
 <br />
-the harvest was performed according to standard protocol:
+The virus harvest was performed according to standard protocol:
 * 3 Plates got transduced
 . Plate I(A is up) completely 10 µg
 {| border="1"
-| 300µl dropped || align="right" |300µl resuspended|| align="right" |450µl resuspended
+| 300µl resuspended|| align="right" |300µl resuspended|| align="right" |450µl resuspended
 |-
-| 600µl dropped||  align="right" |600µl resuspended||  align="right" | control
+| 600µl resuspended||  align="right" |600µl resuspended||  align="right" | control
 |-
 |}
@@ Line 272: / Line 372: @@
 . Plate II(A is up)completely 18 µg
 {| border="1"
-| 300µl dropped || align="right" |300µl resuspended|| align="right" |450µl resuspended
+| 300µl resuspended|| align="right" |300µl resuspended|| align="right" |450µl resuspended
 |-
-| 600µl dropped||  align="right" |600µl resuspended||  align="right" | control
+| 600µl resuspended||  align="right" |600µl resuspended||  align="right" | control
 |-
 |}
@@ Line 281: / Line 381: @@
 . Plate III(A is up) completely 24 µg
 {| border="1"
-| 300µl dropped || align="right" |300µl resuspended|| align="right" |450µl resuspended
+| 300µl resuspended|| align="right" |300µl resuspended|| align="right" |450µl resuspended
 |-
-| 600µl dropped||  align="right" |600µl resuspended||  align="right" | control
+| 600µl resuspended||  align="right" |600µl resuspended||  align="right" | control
 |-
 |}
@@ Line 319: / Line 419: @@
 = Plate 3 B2 : 20,8%       "            from 75,7% <br>
 = Plate 3 B3 : 0,07%       "            from 84,8% <br>
-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
@@ Line 336: / Line 433: @@
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">14.7 Seeding AAV293 for TKGMK constructs   </p>===
+===<p style="font-size:17px; background-color:#00dd77;">14.7 Seeding AAV293 for TKGMK constructs </p>===
 <br />
 <b>The motivation</b><br />
@@ Line 342: / Line 439: @@
 <br /><br />
 <b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">14.7</th>
+<th width="100">15.7</th>
+<th width="100">16.7</th>
+<th width="100">17.7</th>
+<th width="100">18.7</th>
+<th width="100">19.7</th>
+<th width="100">20.7</th>
+<th width="100">21.7</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>FACS</td>
+</tr>
+</table>
 µg Plasmid were used (pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30)<br>
 VIRUS no GOI :pHelper + pAAV_RC<br>
@@ Line 351: / Line 472: @@
 <br />
 <b>The results</b><br />
-Unfortunately the stocks never were used.
+Unfortunately the stocks never were used (exception: see following experiment, 7. august) because later on we noticed that the TKGMK plasmids were not in line with the RFC.
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">7.8 Seeding AAV293 for TKGMK and mVenus stocks </p>===
+===<p style="font-size:17px; background-color:#00dd77;">7.8 Seeding AAV293 for TKGMK and mVenus stocks</p>===
-<b>The motivation</b><br />
+<b>The motivation</b>
-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)..<br />
+After proofing the successfull transduction of tumor cells with the reportergen mVenus the next step is to kill the cells with a prodrug approach. The prodrug of our choice is the guanosine anolog ganciclovir, once activated ganciclovir gets integrated into the growing dna chain which results in termination of dna synthesis and cell death. We transduce the tumor cell line HT1080 with viral particles packed with the thymidine kinase guanosine mono phosphate kinase fusion protein (TKGMK) as gene of interest and the incubation with ganciclovir should kill the cells.<br />
 <br />
 <b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">7.8</th>
+<th width="100">8.8</th>
+<th width="100">9.8</th>
+<th width="100">10.8</th>
+<th width="100">11.8</th>
+<th width="100">12.8</th>
+<th width="100">13.8</th>
+<th width="100">14.8</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>FACS and microscopy</td>
+</tr>
+</table>
+<br />
 {| border="1"
 | Plate  || align="right"  |pAAV_RC/µg|| align="right" |pHelper/µg|| align="right" |GOI/µg
@@ Line 369: / Line 515: @@
 | 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
+| 3||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 TK_GMK clone1 (P54)
 |-
-| 4||  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 (P54)
 |-
-| 5||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 TK_GMK clone2
+| 5||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 TK_GMK clone2 (P55)
 |-
 | 6||  align="right" |3,3 ||  align="right" | 3,3||  align="right" | 3,3 YFP
@@ Line 386: / Line 532: @@
 |-
 |}
-The transduction was performed according to standard protocol.
+<br />The transduction was performed according to standard protocol.
-Platte 1:<br />
+Plate 1:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 411: / Line 557: @@
 </table>
 <br />
-Platte 2:<br />
+Plate 2:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 434: / Line 580: @@
 </table>
 <br />
-Platte 3:<br />
+Plate 3:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 457: / Line 603: @@
 </table>
 <br />
-Platte 4:<br />
+Plate 4:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 480: / Line 626: @@
 </table>
 <br />
-Platte 5:<br />
+Plate 5:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 503: / Line 649: @@
 </table>
 <br />
-Platte 6:<br />
+Plate 6:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 526: / Line 672: @@
 </table>
 <br />
-Platte 7:<br />
+Plate 7:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 549: / Line 695: @@
 </table>
 <br />
-Platte 8:<br />
+Plate 8:<br />
 <table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 590: / Line 736: @@
 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;"><b>11.8 Transduction of HT1080 with no AAV, AAV without GOI and AAV with 10µg</b></p>===
+===<p style="font-size:17px; background-color:#00dd77;"><b>14.8 Seeding different amounts of AAV293 for transfection</b></p>===
-<b>The motivation</b>: 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).<br />
-<b>The plan</b>
-<br />
-Plate I
-{| border="1"
-| control, no cells || align="right" |no GOI 150µl|| align="right" |10µg 500µl
-|-
-| control, no virus||  align="right" |no GOI 300µl||  align="right" | 10µg 750µl
-|-
-|}
-Plate II
-{| border="1"
-| control, no cells || align="right" |no GOI 150µl|| align="right" |10µg 500µl
-|-
-| control, no virus||  align="right" |no GOI 300µl||  align="right" | 10µg 750µl
-|-
-|}
-Plate III
-{| border="1"
-| control, no cells || align="right" |no GOI 150µl|| align="right" |10µg 500µl
-|-
-| control, no virus||  align="right" |no virus||  align="right" | 10µg 750µl
-|-
-|}
-<b>The results</b>
-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!
-===<p style="font-size:17px; background-color:#00dd77;"><b>14.8 Seeding different amounts of AAV293 for transfection </b></p>===
 <b>The motivation</b><br />
 We want to know which confluence of AAV293 cells is optimal for AAV production.<br />
 <br />
 <b>The plan</b>
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">14.8</th>
+<th width="100">15.8</th>
+<th width="100">16.8</th>
+<th width="100">17.8</th>
+<th width="100">18.8</th>
+<th width="100">19.8</th>
+<th width="100">20.8</th>
+<th width="100">21.8</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>FACS</td>
+</tr>
+</table>
 <br />
 We seed different amounts of cells in 10 cm<sup>2</sup>, and check the highest titer.<br />
 <br />
 {| border="1"
-| Plate  || align="right"  |amount of cells
+| Plate  || align="right"  |amount of seeded AAV-293 cells
 |-
 | 1||  align="right" |100 000
@@ Line 654: / Line 793: @@
 |}
-The cells were transfected with 40µg YFP, 3,3µg Rep/Cap and 3,3µg pHelper.
+The cells were transfected with 40µg standard CMV_YFP, 3,3µg Rep/Cap and 3,3µg pHelper.
 Transduction was performed according to standard protocol.
@@ Line 771: / Line 910: @@
 </table>
 <b>The results</b><br />
+The FACS was still not available but we could easily detect a significant higher YFP expression (70-80%) at the following positions:
+* Plate 1: A2 and B2, A3 and B3
+* Plate 2: A2 and B2, A3 and B3
+Therefore we conclude that we have to seed between 100000 and 500000 cells for optimal virus production precedure.
-===<p style="font-size:17px; background-color:#00dd77;">17.8 transduction with viral stocks 20 µg and 40 µg</p>===
+===<p style="font-size:17px; background-color:#00dd77;">17.8 transduction with viral stocks 20 µg and 40 µg GOI-plasmid</p>===
+<b>The Motivation</b><br />
+We want to FACS our stocks to get valid data for further investigation of optimal virus assembly. Therefore created stocks with 20 and 40 µg CMV_mVenus.
+<br /><b>The Plan</b><br />
+<br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">17.8</th>
+<th width="100">18.8</th>
+<th width="100">19.8</th>
+<th width="100">20.8</th>
+<th width="100">21.8</th>
+<th width="100">22.8</th>
+<th width="100">23.8</th>
+<th width="100">24.8</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>FACS</td>
+</tr>
+</table>
+<br />
 <li>1. plate 50.000 cells, YFP
 {| align=right
@@ Line 834: / Line 1,005: @@
 |-
 |}
-<li>5. plate 100.000 cells TKGMK
+<b>The Results</b><br />
-{| align=right
+Although we were told that the FACS would be available in time it wasn't. So the cells could unfortunately not be checked for their YFP expression =(.
-|}
-{| border="1"
-!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)
-|-
-|}
 ===<p style="font-size:17px; background-color:#00dd77;">16.8 Seeding AAV293 for transfection with new R/C</p>===
 <b>The motivation</b> <br />
-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.
+We want examine the functionality of different R/C constructs and the new eGFP gene of interest. This transfection was performed with new AAV-293 cells which were thawed.
 <b>The plan</b> <br />
-* 2x10<sup>6</sup> cells were seeded (instead of 3x10<sup>6</sup>) --> hopefully better virus-production
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">16.8</th>
+<th width="100">17.8</th>
+<th width="100">18.8</th>
+<th width="100">19.8</th>
+<th width="100">20.8</th>
+<th width="100">21.8</th>
+<th width="100">22.8</th>
+<th width="100">23.8</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>FACS</td>
+</tr>
+</table>
+* 2x10<sup>6</sup> cells were seeded
 * used plasmids:
 **P50   c= 429 ng/µl (RC)
@@ Line 873: / Line 1,050: @@
 The transfection was performed according to standard protocol.
-* <b> 1. plate:</b> Lipo-Transfection
+* <b> 1. plate:</b> Lipo-Transfection => named Lipo
-* <b> 2. plate:</b> Lipo-Transfection
+* <b> 2. plate:</b> Lipo-Transfection => named Lipo
-* <b> 3. plate:</b> 3,3 µg (RC), 3,3 µg (pHelper), 3,3 µg (YFP)
+* <b> 3. plate:</b> 3,3 µg (RC), 3,3 µg (pHelper), 3,3 µg (YFP) => was not used for transduction
-* <b> 4. plate:</b> 10 µg (RC), 10 µg (pHelper), 3,3 µg (YFP)
+* <b> 4. plate:</b> 10 µg (RC), 10 µg (pHelper), 3,3 µg (YFP) => named CaCl<sub>2</sub>
-* <b> 5. plate:</b> 10 µg (RC mutant P158a), 10 µg (pHelper), 3,3 µg (YFP)
+* <b> 5. plate:</b> 10 µg (RC mutant P158a), 10 µg (pHelper), 3,3 µg (YFP)=> CaCl<sub>2</sub> 10 µg RC mutant P158a
-* <b> 6. plate:</b> 10 µg (RC mutant P158b), 10 µg (pHelper), 3,3 µg (YFP)
+* <b> 6. plate:</b> 10 µg (RC mutant P158b), 10 µg (pHelper), 3,3 µg (YFP)=> CaCl<sub>2</sub> 10 µg RC mutant P158b
-* <b> 7. plate:</b> 3,3 µg (RC mutant P158a), 3,3 µg (pHelper), 3,3 µg (YFP)
+* <b> 7. plate:</b> 3,3 µg (RC mutant P158a), 3,3 µg (pHelper), 3,3 µg (YFP)=> CaCl<sub>2</sub> 3,3 µg RC mutant P158a
-* <b> 8. plate:</b> 3,3 µg (RC mutant P158b), 3,3 µg (pHelper), 3,3 µg (YFP)
+* <b> 8. plate:</b> 3,3 µg (RC mutant P158b), 3,3 µg (pHelper), 3,3 µg (YFP)=> CaCl<sub>2</sub> 3,3 µg RC mutant P158b
-* <b> 9. plate:</b> 10 µg (RC), 10 µg (pHelper), 3,3 µg (eGFP)
+* <b> 9. plate:</b> 10 µg (RC), 10 µg (pHelper), 3,3 µg (eGFP)=> CaCl<sub>2</sub> eGFP 3,3 µg
-* <b> 10. plate:</b> 10 µg (RC), 10 µg (pHelper), 10 µg (eGFP)
+* <b> 10. plate:</b> 10 µg (RC), 10 µg (pHelper), 10 µg (eGFP)=> CaCl<sub>2</sub> eGFP 10 µg
-* <b> 11. plate:</b> 10 µg (RC), 10 µg (pHelper), 20 µg (eGFP)
+* <b> 11. plate:</b> 10 µg (RC), 10 µg (pHelper), 20 µg (eGFP)=> CaCl<sub>2</sub> eGFP 20 µg
-* <b> 12. plate:</b> 10 µg (RC), 10 µg (pHelper), 40 µg (eGFP)
+* <b> 12. plate:</b> 10 µg (RC), 10 µg (pHelper), 40 µg (eGFP)=> CaCl<sub>2</sub> eGFP 40 µg
 <b>The results</b> <br />
@@ Line 890: / Line 1,067: @@
 [[Image:Freiburg10 23.8.2010 FACS eGFP Lipofection 200.000.jpg|800px]]
-As you can see the eGFP constructs very well. And even the transduction efficiency of the standard R/C is doubled.
+As you can see the eGFP constructs worked very well. And even the transduction efficiency of the standard R/C is ok, the conclusion is that in previous transfections the AAV293 cells were to confluent so their transfection capability. The confluence of the cells is crucial! It also seems that our used amounts of plasmids had no decisive effect on the YFP expression because of the obviously variating results. A final conclusion is still due.
-===<p style="font-size:17px; background-color:#00dd77;">20.8</p>===
-<b>The Motivation</b><br />
-Seeding HT1080 for transfection with viral stocks from 18.8
-<br />
-<b>The Plan</b><br />
-We harvest <b>four</b> T75 flasks and seed them into 6er dishes (200.000 cells in each well)
-<br />
-<b>the Results</b><br />
-<br />
-<br />The Facs data at 22.8
 ===<p style="font-size:17px; background-color:#00dd77;">19.8 Seeding AAv293 for transfection with different HBS buffers </p>===
@@ Line 910: / Line 1,075: @@
 We want to try different 2xHBS Buffers (pH) and their influence on transfection efficiency!<br /><br />
 <b>The Plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">19.8</th>
+<th width="100">20.8</th>
+<th width="100">21.8</th>
+<th width="100">22.8</th>
+<th width="100">23.8</th>
+<th width="100">24.8</th>
+<th width="100">25.8</th>
+<th width="100">26.8</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td></td>
+<td>FACS</td>
+</tr>
+</table>
 We try 5 different 2xHBS buffers:
@@ Line 921: / Line 1,110: @@
 <br />
-Used Plasmids: Standard protocol: 10µg of each plasmid
+Used Plasmids: 10µg of each plasmid
 <ul>
 <li>GOI: mVENUS => P262, conc:1148ng/µl; used amount: 8,71µl</li>
@@ Line 928: / Line 1,117: @@
 <li>RepCap => 10.8: 1348ng/µl; used amount: 7,41µl</li>
 </ul>
-<br />
-Eight new viral stocks were made with pHelper:20µl and RepCap:7,41µl each:<br />
-<ol>
-<li>mVenus:8,71µl; pH of 2xHBS:7,06</li>
-<li>mVenus:8,71µl; pH of 2xHBS:7,08 </li>
-<li>mVenus:8,71µl; pH of 2xHBS:7,10 </li>
-<li>mVenus:8,71µl; pH of 2xHBS:7,12 </li>
-<li>mVenus:8,71µl; pH of 2xHBS:7,14 </li>
-<li>TKGMK:6,67µl; pH of 2xHBS:7,08</li>
-<li>TKGMK:6,67µl; pH of 2xHBS:7,10</li>
-<li>TKGMK:6,67µl; pH of 2xHBS:7,12</li>
-</ol>
 <br />
 *Transduction of three 6-well plates:
@@ Line 1,013: / Line 1,190: @@
 *(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
+*(3)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,112
 *(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 (<b>eGFP</b>)
 <br />
 <b>the Results</b><br />
 AAV-Harvesting is at 24.8, same day Transduction and FACS will be done at 26.8
-[[Image:Freiburg10 26.8.2010 FACES different 2xHBS buffers.jpg|800px]]
+[[Image:Freiburg10 CC 26.8 different HBS.jpg|800px]]
-As you can see the 2xHBS with pH 4.10 performed best, in future experiments this buffer will be used.
+As you can see the 2xHBS with pH 7.112 performed best, in future experiments this buffer will be used.
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">XXXXXXXXXXXXXXXXXXXXXXXXXX </p>===
+===<p style="font-size:17px; background-color:#00dd77;">22.8: Transfection of AAV293 with different amounts of cells, and two plasmid concentrations</p>===
-<h3>Checking transduction efficiency via fluorescence microscopy</h3>
-<b>the motivation</b><br />
-<br />
-The transduction efficiency from the viral stocks which were created with different amounts of AAV293 was checked.<br />
-<b>The plan</b><br />
-<br />
-<ol>
-<li>plate 1:</li>
-<ul><li>stock 1 (with 100.000 cells) </li>
-<li>stock 2 (with 200.000 cells) </li>
- </ul>
-<li>plate 2:</li>
-<ul><li>stock 3 (with 400.000 cells) </li>
-<li>stock 4 (with 500.000 cells) </li>
-</ul>
-<li>plate 3:</li>
-<ul><li>stock 5 (with 800.000 cells) </li>
-<li>stock 6 (with 1.000.000 cells) </li>
-</ul>
-<li>plate 4:</li>
-<ul><li>stock 7 (with 1.200.000 cells)  </li>
-<li>stock 8 (with 1.500.000 cells)  </li>
-</ul>
-<li>plate 5:</li>
-<ul><li>stock 9 (with 1.750.000 cells)  </li>
-<li>stock 10 (with 1.750.000 cells)  </li>
-</ul>
-</ol>
-<b>The results</b><br />
-<br />
-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
-===<p style="font-size:17px; background-color:#00dd77;">25.8: Transfection of AAV293 with different amounts of cells, and two plasmid concentrations</p>===
 <b>The motivation</b><br />
 <br />
@@ Line 1,065: / Line 1,208: @@
 <br />
 <b>The plan</b><br />
-<br />
-<ol>
-<li>100.000</li>
-<li>100.000</li>
-<li>200.000</li>
-<li>200.000</li>
-<li>300.000</li>
-<li>300.000</li>
-<li>400.000</li>
-<li>400.000</li>
-<li>500.000</li>
-<li>500.000</li>
-<li>600.000</li>
-<li>600.000</li>
-<li>700.000</li>
-<li>700.000</li>
-</ol>
-<br />
-µg RC P158a four times mutant, 10µg pHelper, 3,3 µg mVenus P162  were pipetted on plates: 2, 4, 6, 8, 10 , 12, 14
-<br />
-,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
-<br />
-<br />
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 1,115: / Line 1,235: @@
 </tr>
 </table>
+The following amount of cells were seeded in 10 cm<sup>2</sup>:
+<ol>
+<li>100.000</li>
+<li>100.000</li>
+<li>200.000</li>
+<li>200.000</li>
+<li>300.000</li>
+<li>300.000</li>
+<li>400.000</li>
+<li>400.000</li>
+<li>500.000</li>
+<li>500.000</li>
+<li>600.000</li>
+<li>600.000</li>
+<li>700.000</li>
+<li>700.000</li>
+</ol>
+<br />
+µg RC P158a four times mutant, 10µg pHelper, 3,3 µg mVenus P162  were pipetted on plates: 2, 4, 6, 8, 10 , 12, 14
+<br />
+,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
+<br />
 <b>The results</b><br />
@@ Line 1,120: / Line 1,264: @@
 [[Image:Freiburg10 30.2010 FACS number of Cells and Beas construct.jpg|800px]]
 <br />
+<br />
-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!
+There is only one value for each approach but nevertheless the average YFP expression should be higher.
+We really dont know the reason for the quite low YFP expression. There are also very good news: the reassembled construct (pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR) created from the single Biobricks works as "well" as the AAV2 with CMV_mVenus!
 <br/>
@@ Line 1,127: / Line 1,272: @@
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">30.8 Seeding AAV293 for 10xTransfection</p>===
+===<p style="font-size:17px; background-color:#00dd77;">30.8 Seeding AAV293 for Transfection of 10 cm<sup>2</sup> plates</p>===
 <br />
 <b>The motivation</b><br />
 We want to create ten hypothetical identical viral stocks to estimate the standard deviation.
-<br />
+<br /><br />
-<b>The plan</b><br />
+<b>The plan</b>
-<br />
 <br />
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
@@ Line 1,169: / Line 1,312: @@
 </ul>
 <br />
-,5 ml of each stock become deepfreeze in.<br />
+,5 ml of each stock be stored at -80°C.<br />
 <br />
-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.
+,5 ml of each stock will be used for Transduction. <br>
-===<p style="font-size:17px; background-color:#00dd77;">XXXXXXX getting valide data of 10µg samples</p>===
+<br>
+Result:
+<br>
+[[Image:Freiburg10_Unbenannt.jpg]]
+<br>
+Standard deviation: about +/- 4,98% YFP expression. All future AAV2 stocks will be pooled if produced with this same plasmids and amounts of plasmids. Unfortunately the YFP expression is still located at around 50%. We would like to have a YFP expression of at least 70% because as soon as we start to test our not yet ready modified AAV2 we suggest the YFP expression will obviously decrease.
-<br />
-<b>The motivation</b><br />
-we want to check the YFP expression
-<br />
-<b>The plan</b><br />
-<br />
-<br />
-<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-<tr>
-<th width="100">Days</th>
-<th width="100">30.8</th>
-<th width="100">31.8</th>
-<th width="100">1.9</th>
-<th width="100">2.9</th>
-</tr>
-<tr>
-<td>Action</td>
-<td>seeding HT</td>
-<td>Transduction</td>
-<td>-</td>
-<td>FACS</td>
-</tr>
-</table>
-<br />
-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.<br />
-<br />
-<b>the results</b>
-===<p style="font-size:17px; background-color:#00dd77;">28.8 Seeding AAV293 for checking functionality of RC vector</p>===
-<b>The motivation</b><br />
-We want to check different R/C constructs, additionally we check if the amount of viral.!!!!!!!!!!!!!!!!!<br />
-<br />
-<b>The plan</b><br />
-<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-<tr>
-<th width="100">Days</th>
-<th width="100">28.8</th>
-<th width="100">29.8</th>
-<th width="100">30.8</th>
-<th width="100">31.9</th>
-<th width="100">1.9</th>
-<th width="100">2.9</th>
-<th width="100">3.9</th>
-<th width="100">4.9</th>
-<th width="100">5.9</th>
-<th width="100">6.9</th>
-</tr>
-<tr>
-<td>Action</td>
-<td>seeding AAV293</td>
-<td>-</td>
-<td>-</td>
-<td>Transfection</td>
-<td>-</td>
-<td>-</td>
-<td>Harvest+Seeding HT</td>
-<td>Transduction</td>
-<td>-</td>
-<td>FACS</td>
-</tr>
-</table>
-<b></b>
-<br />
-Transfection was done with:
-<ul>
-<li>RepCap:P326 => 2481 ng/µl used amount: 4µl => 10µg</li>
-<li>RepCap:P325 => 2051 ng/µl used amount: 4,87µl => 10µg</li>
-<li>pHelper:P356 => 684 ng/µl used amount: 414,62µl => 10µg </li>
-<li>mVenus:P262 => 1148 ng/µl used amount: 8,7µl => 10µg </li>
-</ul>
+===<p style="font-size:17px; background-color:#00dd77;">4.9 Seeding cells for checking constructs (GOIs) with/without HGH and beta globin and reassembled as control </p>===
 <br />
-<b>The results</b><br />
+<b>The motivation</b><br />
-<br />[[Image:Freiburg10 CC 17.9 FACS without HGH beta globin P326 P325 data-CHART.jpg|800px]]
+We want to check the functionality of these genes of interest
-===<p style="font-size:17px; background-color:#00dd77;">4.9 Seeding cells for checking constructs (GOIs) with/without HGH and beta globin and reassembled as control</p>===
-<br />
-<b>The motivation</b><br />
-We want to check if these reassmbled constructs (GOIs) still work!
-<ul>
-<li>P269 lITR_CMV_beta-globin_mVenus_HGH_rITR = 325 ng/µl => 9,66 µl </li>
-<li>P270 lITR_CMV_beta-globin_mVenus_HGH_rITR = 561 ng/µl => 7,26 µl</li>
-<li>P377 lITR_CMV_mVenus_HGH_rITR = 325,04 ng/µl => 30,76 µl   </li>
-<li>P378 lITR_CMV_beta-globin_mVenus_rITR = 561 ng/µl => 17,8µl  </li>
-</ul>
-All constructs are completed with p50 R/C =>> 26,45 µl and pHELPER 14,61µl<br />
-<br />
 <b>The plan</b><br />
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
@@ Line 1,290: / Line 1,357: @@
 </tr>
 </table>
-<b></b>
+<ul>
+<li>P269 pSB1C3_lITR_CMV_beta-globin_mVenus_HGH_rITR = 325 ng/µl => 9,66 µl </li>
+<li>P270 pSB1C3_lITR_CMV_beta-globin_mVenus_HGH_rITR = 561 ng/µl => 7,26 µl</li>
+<li>P377 pSB1C3_lITR_CMV_mVenus_HGH_rITR = 325,04 ng/µl => 30,76 µl   </li>
+<li>P378 pSB1C3_lITR_CMV_beta-globin_mVenus_rITR = 561 ng/µl => 17,8µl  </li>
+</ul>
+Additionally P50: pAAV_RC R/C (26,45 µl) and pHELPER (14,61µl) pipetted to each approach. <br />
+<br />
 <br />
 <b>The results</b><br />
@@ Line 1,298: / Line 1,372: @@
 <br />
 <b>The motivation</b><br />
-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.
+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.
+<br />
 <br />
 <b>The plan</b><br />
-<br />
-<br />
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 1,332: / Line 1,404: @@
 <br />
 <b>the results</b>
+<br />
 <br />
 [[Image:Freiburg10 CC 6.9 Checking Ganciclovir concentrations-CHART.jpg|800px]]
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">15.9_RC_Tryout_compositions</p>===
 <br />
-<b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
 <br />
-<b>The plan</b><br />
+As expected the highest concentration of ganciclovir worked the best but it is remarkable that there is no significant difference between the first three ganciclovir amounts. The most remarkable note is, that in future experiments the incubation time has to be extended to 3 or 4 days.
-In this section, the design of the experiment is explained.
-<br />
-<b>The results</b><br />
-Interpretation and discussion of our results.
-<br />
-===<p style="font-size:17px; background-color:#00dd77;">19.9_Checking_P467_P468</p>===
+===<p style="font-size:17px; background-color:#00dd77;">15.9 Seeding AAV293 for testing our Affibody and other VP-mVenus constructs</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+The pCerulean_VP1up_mVenus_Vp2/3 Construct will be tested in three different compositions.<br />
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+We want to test these constructs:
+<ul>
+<li>pCerulean_VP1up_NLS_Affibody_VP2/3</li>
+<li>pCerulean_VP1up_His_VP2/3</li>
+<li>pCerulean_VP1up_mVenus_Vp2/3</li>
+</ul>
+The His-tag is for purify our constructs and in this case we want to test, if the particles are still infectious. Construct with the affibody will be tested for functionality and even for their better affinity to our EGFR overexpressing cell line A431.
 <br />
 <b>The results</b><br />
 Interpretation and discussion of our results.
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">19.9 Seed AAV293 for testing pTERT promoter</p>===
+===<p style="font-size:17px; background-color:#00dd77;">19.9 Seeding AAV293 for checking two different Rep/Cap Constructs</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+During the project 22 silent nucleotide exchange mutations were introduced into the capsid coding construct to make it compatible with the RFC standards and to have single cutting restriction enzymes flanking the 453 and the 587 loop sequence. Two point mutations had to be dismissed, because either a first test transduction showed that the construct was not working anymore or the insertion of the synthesized gene posed serious problems, because the restriction enzyme did not work.
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+Transfection and transduction was performed according to standard protocol.
 <br />
 <b>The results</b><br />
-Interpretation and discussion of our results.
+[[Image:Freiburg10 CC 19.9 FACS Checking functionality of REP CAP.jpg|800px]]
+<br /><br />
+Finally, we have a construct that is shown to produce infectious particles comparable to the current AAV systems and carries 20 point mutations. Now we can announce that the Adeno-associated Virus is compatible to the RFC standard and the idea to replace the loop sequences via ViralBricks works!
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">20.9 Seeding AAV293 for creating first VP-mVenus-fusion constructs</p>===
+===<p style="font-size:17px; background-color:#00dd77;">22.9 Seeding AAV293 for transfection with different mVenus constructs for checking specific promoter activity</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+We want to check if the CMV and pTERT promoters work equaly in the AAV293, A431 and HT1080 cells. So we transfect them via standard transfection CaCl<sub>2</sub>. It is obvious that the individual cell lines are not equaliy competent to transfection (the AAV293 are optimised for transfection). It seems that this promoter is not that active in AAV293 cells.
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+We transfected the three cell lines A431, AAV293 and HT1080 with following three constructs:
-<br />
+<ul>
-<b>The results</b><br />
+<li>CMV_mVenus</li>
-Interpretation and discussion of our results.
+<li>CMV_eGFP</li>
+<li>pTERT_mVenus</li>
+</ul>
 <br />
+Remember, that mVenus and eGFP have the exact same excitation wavelenght. We took the pictures with the same parameters, but saved the data in the wrong formate (.tif instead of .zvi), <b>so we werent able to change the YFP expression from green to yellow</b>.
-===<p style="font-size:17px; background-color:#00dd77;">22.9 Transfection with Volkers R/C constructs</p>===
 <br />
-<b>The motivation</b>
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
-We want to check if these R/C still work! <br />
-<br /><b>The plan</b><br />
-<br /><table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
 <th width="100">Days</th>
-<th width="100">20.9</th>
-<th width="100">21.9</th>
 <th width="100">22.9</th>
 <th width="100">23.9</th>
-<th width="100">24.9</th>
+<th width="100">24.10</th>
-<th width="100">25.9</th>
+<th width="100">25.10</th>
-<th width="100">26.9</th>
+<th width="100">26.10</th>
-<th width="100">27.9</th>
-<th width="100">28.9</th>
 </tr>
 <tr>
 <td>Action</td>
-<td>seeding AAV293: 4*6wells</td>
+<td>seeding AAV293</td>
 <td>-</td>
-<td>Transfection: 4 constructs</td>
+<td>Transfection</td>
-<td>-</td>
+<td>fluorescence microscopy</td>
-<td>-</td>
+<td>fluorescence microscopy</td>
-<td>Seeding HT</td>
-<td>Harvest + Transduction</td>
-<td>-</td>
-<td>FACS</td>
 </tr>
-</table><br />
+</table>
-<b>The results</b><br />
 <br />
-On the 23.9 it was shure that something went wrong with the nomenclature, so this approach is definitely not working => threwn away!
+<gallery widths=300px heights=300px perrow=2 caption="Transfection of different cell types with different promoter_reportergenes">
+Image:Freiburg10 CC AAV293 16h eGFP STANDARD from Sven.jpg|AAV293 CMV_eGFP 16h post transfection
+Image:Freiburg10 CC AAV293 16h mVENUS reassembled1.jpg|AAV293 CMV_mVenus 16h post transfection
+Image:Freiburg10 CC AAV293 16h pTERT mVenus1.jpg|AAV293 pTERT_mVenus 16h post transfection
-===<p style="font-size:17px; background-color:#00dd77;">22.9 Seeding AAV293 for transfection with different mVenus constructs for checking specific promoter activity</p>===
+Image:Freiburg10 A293 48h pTERT construct1 low light.jpg|AAV293 CMV_eGFP 48h post transfection
-<br />
+Image:Freiburg10 48h HT1080 reassembled construct1.jpg|HT1080 CMV_mVenus 48h post transfection
-<b>The motivation</b><br />
+Image:Freiburg10_48h_A431_reassembled_construct1.jpg|A431 CMV_mVenus 48h post transfection
-This short introduction provides an inside into our thoughts for this experiment.
+</gallery>
-<br />
-<b>The plan</b><br />
-In this section, the design of the experiment is explained.
 <br />
 <b>The results</b><br />
-Interpretation and discussion of our results.
+As excepted, the expression of the CMV_mVenus and CMV_eGFP was very high even at 16h post transfection. The pTERT_mVenus construct worked aswell, but significantly weaker!
+Problematic is the fact, that there was no expression in HT1080 and A431 detectable. Due the fact that these cell lines are not optimized for CaCl<sub>2</sub> transfection. We can't evaluate if the bad transfection efficiency or the low promoter activity of pTERT are responsible for the results.
 <br />
@@ Line 1,438: / Line 1,502: @@
 <br />
 <b>The motivation</b>
-<br /> Test the 18 constructs for functionality on A431 and HT1080 cells.<br />
+<br /> Test the following 18 constructs for functionality on A431 and HT1080 cells. Each of these constructs getting transfected in different compositions 25%, 50%, 75% and 90% with VP2_knockout 75%. In this way we are able to create viral particles with this ratios of VP proteins. We hope that the created particles are still infectious.<br />
+<ul>
+<li>pCerulean_CFP_MiddleLinker_VP2/3insCap P528</li>
+<li>pCerulean_Zegfr:1907_ShortlLinker_VP2/3insCap P527</li>
+<li>pCerulean_6xHis_Middlelinker_VP2/3insCap P534</li>
+<li>pCerulean_Zegfr:1907_Middlelinker_VP2/3insCap P535</li>
+<li>pCerulean_Zegfr:1907_SEG_VP2/3_Capins 10:90 P502</li>
+<li>pCerulean_Zegfr:1907_LongLinker_VP2/3_CapIns P503</li>
+</ul>
+<br />
 <b>The plan</b><br />
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
@@ Line 1,471: / Line 1,544: @@
 <br> [[Image:Freiburg 10 FACS Linker Libary.jpg|800px]]
 <br />
+Obviously the constructs still work!
 ===<p style="font-size:17px; background-color:#00dd77;">22.9 Seeding cells for testing pTERT_mVenus in R/C P326 and P431 </p>===
 <br />
-<b>The motivation</b>
+<b>The motivation</b><br />
-We want to check if the pTERT-promoter is realiable for tumor specific gene expression. <br /><br />
+We want to check if the pTERT-promoter is realiable for tumor specific gene expression, we use two different kinds of R/C. <br /><br />
 <b>The plan</b><br />
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
@@ Line 1,511: / Line 1,585: @@
 </tr>
 </table>
+<br />
+<b>The results</b>
 <br />
+===<p style="font-size:17px; background-color:#00dd77;">29.9 Checking location of VP proteins via fluorescence microscopy and checking fractions of centrifugation steps via fluorescence spectrometer</p>===
 <br />
-<b>The results</b>
+<b>The motivation</b>: We want to figure out if it is possible to purify our viral stocks via centrifugation with 10.000 g and/or 20.000 g. In theory/ according to the literature the viral particles should be in the cells and attached to the HSPGs at the cellular surfaces.
 <br />
+<b>The plan</b>: Two different viral stocks were prepared:
+<br /><br />
+Stock 1 with VP2-mVenus-fusion-Capsid loaded with TKGMK (4 wells from a 6 well plate)<br />
+<ul>
+<li>R/C 1: 50% pCerulean_VP1up_NLS_mVenus_Vp2/3 P501</li>
+<li>R/C 2: 50% Rep/Cap2: P449 pAAV_RC_4fachmut_VP1-ko:</li>
+<li>GOI: TKGMK: P82.b</li>
+<li>pHelper</li>
+</ul>
+<br />
+Stock 2 "standard" virus (4 wells from a 6 well plate)<br />
+<ul>
+<li>R/C 1 P486</li>
+<li>GOI: TKGMK: P82.b</li>
+<li>pHelper</li>
+</ul>
+These two stocks got harvested according to standard protocol (scratching cells, transfer them in to 15 ml falcons, resuling 12ml solution total). After centrifugation (10 min 200g), the supernatant got transferred into two new 15 ml falcons and the pellets were resuspended with 10 ml DMEM Medium. The Stocks got freezed thawed two times so finally there were:
+<ol>
+<li>DMEM as negative control</li>
+<li>resuspended Pellet with mVenus-fusioned viral capsids</li>
+<li>resuspended Pellet with "standard virus"</li>
+<li>supernatant with mVenus-fusioned viral capsids</li>
+<li>supernatant with "standard virus"</li>
+</ol>
+<br />
+The fluorescence of each stock was measured via spectrometer. After that, stocks 2 and 4 were centrifugated 10min with 10.000 g followed by an other fluorescence measuring. Finally the stocks got spin down with 20.000 g for 10 min
+<br />
+<b>The results</b>:
+<br />
+<gallery widths=300px heights=300px perrow=2 caption="Transfection with VP-mVenus-fusion constructs">
+Image:Freiburg10 VP mVenus fusion localisation 1.10 Close Picture2.jpg|Picture two days post transfection.
+</gallery>
-===<p style="font-size:17px; background-color:#00dd77;">28.9 Seeding cells for testing loop insertions </p>===
 <br />
-<b>The motivation</b><br />
+[[Image: Freiburg10 mVenus VP fusion fluorescence centrifugation experiment graph.jpg|thumb|center|800px]]
 <br />
-<b>The plan</b><br />
+[[Image:Freiburg10 mVenus VP fusion fluorescence centrifugation experiment 526nm.jpg|thumb|center|800px]]
+<br /><b>The conclusions</b>:
+The highest amount of fluorescence was measured in the supernatant, the individual centrifugation steps had <b>no decrease in fluorescence</b> as consequence!
 <br />
+Keep in mind, without further investigation it is not valid to say that we can purify our stocks via 10-20.000 g centrifugation steps, because we dont know if the viral capsids are still intact! The FACS analysis are not sufficient enough to make a decision, because our last samples had YFP as GOI (=> so efficient cell sorting was not possible). We need to do qPCRs to make a valid evidence.
+<br />
+<br />
+===<p style="font-size:17px; background-color:#00dd77;">30.9 Production of new Standard mVenus Vector</p>===
+<br />
+<b>The motivation</b><br />
+The AAV2 with 4 point mutations, inserted rep and cap and remutated KpnI restriction has to be checked for its functionality in order to compare it with the wild type AAV2. Hopefully there will be no significant difference.<br />
+<b>The plan</b><br />
+After the "delay" 200.000 A431 and HT1080 cells were seeded and transduced on the following day with 0,5 ml of this AAV2 mutant.
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
 <th width="100">Days</th>
-<th width="100">28.9</th>
-<th width="100">29.9</th>
 <th width="100">30.9</th>
 <th width="100">1.10</th>
@@ Line 1,536: / Line 1,655: @@
 <th width="100">5.10</th>
 <th width="100">6.10</th>
+<th width="100">7.10</th>
+<th width="100">8.10</th>
 </tr>
 <tr>
@@ Line 1,545: / Line 1,666: @@
 <td>-</td>
 <td>Seeding HT</td>
-<td>Harvest + Transduction</td>
+<td>Harvest</td>
-<td>-</td>
+<td>delayed because of cell death</td>
-<td>FACS</td>
+<td>delayed because of cell death</td>
 </tr>
 </table>
-<b>The results</b>
+<br />
+<b>The results</b><br />
+The stocks were harvested successfully
+<br />
+===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for production of standard TKGMK vector  </p>===
-===<p style="font-size:17px; background-color:#00dd77;">29.9 Checking location of VP proteins via fluorescence microscopy</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+We want to create a big amount of viral particles with TKGMK as gene of interest for using it as an standard vector for the MTT-assay.
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
 <th width="100">Days</th>
-<th width="100">29.9</th>
 <th width="100">30.9</th>
 <th width="100">1.10</th>
@@ Line 1,574: / Line 1,696: @@
 <th width="100">6.10</th>
 <th width="100">7.10</th>
+<th width="100">8.10</th>
 </tr>
 <tr>
@@ Line 1,583: / Line 1,706: @@
 <td>-</td>
 <td>Seeding HT</td>
-<td>Harvest + Transduction</td>
+<td>Harvest</td>
-<td>-</td>
+<td>delayed because of cell death</td>
-<td>FACS</td>
+<td>delayed because of cell death</td>
 </tr>
 </table>
 <br />
 <b>The results</b><br />
-Interpretation and discussion of our results.
+The viral stocks were harvested efficiently and are ready to use.
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">30.9_Production_of_Standard_mVenus_Vector</p>===
+===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for production of vectors with mVenus missing beta globin</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+Beta globin is said to improve (stratagene) the expression so we expect a worse mVenus (reportergene) expression compared to the standard AAVs. Therefore the seeded A431 and HT1080 cells were transduced with a AAV2 mutant missing beta-globin.
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+Ater the "delay" 200.000 HT1080 and A431 cells were seeded and transduced with 0,5 ml AAV2 mutant on the following day.
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 1,621: / Line 1,744: @@
 <td>-</td>
 <td>Seeding HT</td>
-<td>Harvest + Transduction</td>
+<td>Harvest</td>
-<td>-</td>
+<td>delayed because of cell death</td>
-<td>FACS</td>
+<td>delayed because of cell death</td>
 </tr>
 </table>
@@ Line 1,631: / Line 1,754: @@
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for production of standard TKGMK vector  </p>===
+===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for production of vectors with mVenus missing HGH</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+HGH is responsible for the mRNA polyadenylation of the virus so we expect expect a worse mVenus (reportergene) expression compared to the standard AAVs. Therefore the seeded A431 and HT1080 cells were transduced with a AAV2 mutant missing HGH.
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+After the "delay" 200.000 A431 and HT1080 cells were seeded and transduced on the following day with 0,5 ml AAV2 mutant missing HGH.
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 1,659: / Line 1,782: @@
 <td>-</td>
 <td>Seeding HT</td>
-<td>Harvest + Transduction</td>
+<td>Harvest</td>
 <td>delayed because of cell death</td>
 <td>delayed because of cell death</td>
@@ Line 1,669: / Line 1,792: @@
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for production of vectors with mVenus missing beta globin</p>===
+===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for testing RC with HSPG knockout for functionality</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+The primary AAV2 receptor (the HSPG receptor) was knocked out so we expect a worse tranduction rate (about 0,3 to 0,4 of the standard transduction rate, assuming that there are HSPGs on the HT1080 and A431 cell surface). The worse the better.
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+After the "delay" 200.000 A431 and HT1080 cells were seeded and transduced on the following day with 0,5 ml AAV2 mutant with a HSPG-receptor knockout.
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 1,704: / Line 1,827: @@
 <br />
 <b>The results</b><br />
-Interpretation and discussion of our results.
+[[Image:Freiburg10 HT1080 missing HSPG binding motif.png|thumb|center|800px]]
+[[Image:Freiburg10 A431 missing HSPG binding motif.png|thumb|center|800px]]
 <br />
+There is a significant difference in transduction efficiency. The actual reduction is even higher than expected, according to literature it should be around 60%, we reached 80%. We measured via qPCR the amount of viral particles for each stock, and they are nearly the same the HSPG Knockout has 2*10<sup>7</sup>particles per ml , the stock with HSPG-binding motif has 4.5*10<sup>6</sup> particles per ml.
-===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for production of vectors with mVenus missing HGH</p>===
+===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for testing RC WITHOUT HSPG knockout for functionality</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+The produced vector has the intact HSPG-binding motif, so we can compare it with the vector produced in: "Seeding AAV293 for testing RC with HSPG knockout for functionality"
 <br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+Transfection and transduction were performed according to standard protocol.
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
@@ Line 1,742: / Line 1,867: @@
 <br />
 <b>The results</b><br />
-Interpretation and discussion of our results.
+See 30.9 Seeding AAV293 for testing RC with HSPG knockout for functionality
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for testing RC with HSPG knockout for functionality</p>===
+===<p style="font-size:17px; background-color:#00dd77;">12.10 Seeding AAV293 cells for transfection with VP-BAP</p>===
 <br />
-<b>The motivation</b><br />
+<b>The motivation</b>
-This short introduction provides an inside into our thoughts for this experiment.
 <br />
+We want to create a viral stock for purification and biotinylation, the ratio of the Rep/Cap plasmids is 1:1.<br />
 <b>The plan</b><br />
-In this section, the design of the experiment is explained.
+Transfection was performed according to standard protocol:
+<ul>
+<li>Gene of interest: mVenus</li>
+<li>R/C: pSB1C3-001_pCMV_[AAV]VP123_587_BAP in cotransfection with pSB1C3_001_RC_IRCK_P5tataless clone 1</li>
+</ul>
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
 <th width="100">Days</th>
-<th width="100">30.9</th>
+<th width="100">12.10</th>
-<th width="100">1.10</th>
+<th width="100">13.10</th>
-<th width="100">2.10</th>
+<th width="100">14.10</th>
-<th width="100">3.10</th>
+<th width="100">15.10</th>
-<th width="100">4.10</th>
+<th width="100">16.10</th>
-<th width="100">5.10</th>
+<th width="100">17.10</th>
-<th width="100">6.10</th>
+<th width="100">18.10</th>
-<th width="100">7.10</th>
+<th width="100">19.10</th>
-<th width="100">8.10</th>
+<th width="100">20.10</th>
 </tr>
 <tr>
@@ Line 1,773: / Line 1,903: @@
 <td>-</td>
 <td>Seeding HT</td>
-<td>Harvest</td>
+<td>Harvest + Transduction</td>
-<td>delayed because of cell death</td>
+<td>-</td>
-<td>delayed because of cell death</td>
+<td>FACS</td>
 </tr>
 </table>
+<b>The results</b>
+===<p style="font-size:17px; background-color:#00dd77;">12.10 Seeding AAV293 cells for transfection with mVenus as gene of interest and pCerulean_VP1up_NLS_mVenus_VP2/3_HSPG-KO</p>===
 <br />
-<b>The results</b><br />
+<b>The motivation</b>
-Interpretation and discussion of our results.
 <br />
+We want to create a viral stock for purification and biotinylation, the ratio of the Rep/Cap plasmids is 1:1.<br />
+<b>The plan</b><br />
+Transfection was performed according to standard protocol:
+<ul>
+<li>Gene of interest: mVenus</li>
+<li>R/C: pCerulean_VP1up_NLS_mVenus_VP2/3_HSPG-KO in cotransfection with pSB1C3_001_RC_IRCK_VP1-ko_HSPG-ko_P5tataless cl1</li>
+</ul>
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">12.10</th>
+<th width="100">13.10</th>
+<th width="100">14.10</th>
+<th width="100">15.10</th>
+<th width="100">16.10</th>
+<th width="100">17.10</th>
+<th width="100">18.10</th>
+<th width="100">19.10</th>
+<th width="100">20.10</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td>-</td>
+<td>FACS</td>
+</tr>
+</table>
+<b>The results</b>
-===<p style="font-size:17px; background-color:#00dd77;">30.9 Seeding AAV293 for testing RC WITHOUT HSP knockout for functionality</p>===
+===<p style="font-size:17px; background-color:#00dd77;">12.10 Seeding AAV293 cells for transfection with mVenus as GOI and CFP_Middlelinker_VP2/3_HSPG-KO</p>===
 <br />
 <b>The motivation</b><br />
-This short introduction provides an inside into our thoughts for this experiment.
+This viral stock is for live imaging and probably for western blotting, the ratio of the Rep/Cap plasmids is 1:1.<br />
+<b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">18.10</th>
+<th width="100">19.10</th>
+<th width="100">20.10</th>
+<th width="100">21.10</th>
+<th width="100">22.10</th>
+<th width="100">23.10</th>
+<th width="100">24.10</th>
+<th width="100">25.10</th>
+<th width="100">26.10</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td>-</td>
+<td>FACS</td>
+</tr>
+</table><br />
+Transfection was performed according to standard protocol:
+<ul>
+<li>Gene of interest: mVenus</li>
+<li>R/C: CFP_Middlelinker_VP2/3_HSPG-KO in cotransfection with pSB1C3_001_RC_IRCK_P5tataless clone 1</li>
+<br />
+</ul>
+<b>The results</b>
+===<p style="font-size:17px; background-color:#00dd77;">17.10 Production of AAV2 with 587-His</p>===
+Investigator Patrick <br>
+<br>
+<b>The motivation</b><br />
+We wanted to produce AAV2 which could be purified via affinity purification. Therefore 10 x 10cm^2 were seeded with AAV-293. Five of these plates contained serum-free medium and cells accustomized to this. <br>
+Used plasmids:
+* pHelper
+* pSB1C3_leftITR_CMV_betaglobin_mvenus_hGH_rightITR (P541)
+* 50% pSB1C3_001_RC_IRCK_P5tataless (P712)
+* 50% pSB1C3-001_pCMV_[AAV2]VP123_587_HIS (P638)
 <br />
 <b>The plan</b><br />
 In this section, the design of the experiment is explained.
+<br />
 <table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
 <tr>
 <th width="100">Days</th>
-<th width="100">30.9</th>
+<th width="100">17.10</th>
-<th width="100">1.10</th>
+<th width="100">19.10</th>
-<th width="100">2.10</th>
+<th width="100">20.10</th>
-<th width="100">3.10</th>
+<th width="100">21.10</th>
-<th width="100">4.10</th>
+<th width="100">22.10</th>
-<th width="100">5.10</th>
+<th width="100">23.10</th>
-<th width="100">6.10</th>
+<th width="100">24.10</th>
-<th width="100">7.10</th>
+<th width="100">25.10</th>
-<th width="100">8.10</th>
+<th width="100">26.10</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>Transfection</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>AAV2 harvest</td>
+<td>Transduction</td>
+<td></td>
+<td>FACS</td>
+<td></td>
+</tr>
+</table><br />
+<b>The results</b><br />
+Interpretation and discussion of our results.
+<br />
+===<p style="font-size:17px; background-color:#00dd77;">18.10 Seeding AAV293 cells for transfection Superconstructs</p>===
+<br />
+<b>The motivation</b><br />
+We want to create a viral stock for purification and biotinylation, the ratio of the Rep/Cap plasmids is 1:1.<br />
+<b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">18.10</th>
+<th width="100">19.10</th>
+<th width="100">20.10</th>
+<th width="100">21.10</th>
+<th width="100">22.10</th>
+<th width="100">23.10</th>
+<th width="100">24.10</th>
+<th width="100">25.10</th>
+<th width="100">26.10</th>
 </tr>
 <tr>
@@ Line 1,811: / Line 2,065: @@
 <td>-</td>
 <td>Seeding HT</td>
-<td>Harvest</td>
+<td>Harvest + Transduction</td>
-<td>delayed because of cell death</td>
+<td>-</td>
-<td>delayed because of cell death</td>
+<td>FACS</td>
+</tr>
+</table><br />
+Transfection was performed according to standard protocol:<br />
+<br />
+<b>Gene of interest: CMV_mVenus</b>
+<ul>
+<li>pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis in ratio 1:1 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1</li>
+<li>pSB1C3-001_pCMV_[AAV2]VP123_453_Z34C in ratio 1:1 pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10</li>
+<li>pSB1C3_001_CMV_VP123_587-KO_Z34C in ratio 1:1 pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10</li>
+<li> pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer in ratio 1:1 pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10</li>
+</ul><br />
+<b>Gene of interest: pTERT_TKGMK</b>
+<ul>
+<li>pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis in ratio 1:1 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1</li>
+</ul><br />
+<b>Gene of interest: CMV_TKGMK</b>
+<ul>
+<li>pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis in ratio 1:1 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1</li>
+</ul><br />
+<b>The results</b>
+===<p style="font-size:17px; background-color:#00dd77;">18.10 Seeding AAV293 cells for transfection with Cytosindeaminase</p>===
+<br />
+<b>The motivation</b><br />
+<br />
+We want to create viral stocks with CMV_Cytosindeaminase and pTERT_Cytosindeaminase as gene of interests, to  check qualitatively the bystander effect.<br />
+<br /><b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">18.10</th>
+<th width="100">19.10</th>
+<th width="100">20.10</th>
+<th width="100">21.10</th>
+<th width="100">22.10</th>
+<th width="100">23.10</th>
+<th width="100">24.10</th>
+<th width="100">25.10</th>
+<th width="100">26.10</th>
+<th width="100">27.10</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>seeding AAV293</td>
+<td>-</td>
+<td>-</td>
+<td>Transfection</td>
+<td>-</td>
+<td>-</td>
+<td>Seeding HT</td>
+<td>Harvest + Transduction</td>
+<td>-</td>
+<td>FACS</td>
 </tr>
 </table>
+Transfection, harvest and transduction were performed according to standard protocol:<br />
 <br />
-<b>The results</b><br />
+<b>Gene of interest: CMV_Cytosindeaminase</b>
-Interpretation and discussion of our results.
+<ul>
+<li>P712</li>
+</ul>
+<b>Gene of interest: pTERT_Cytosindeaminase</b>
+<ul>
+<li>P712</li>
+</ul>
 <br />
-===<p style="font-size:17px; background-color:#00dd77;">Experiment</p>===
+<b>The results</b>
+===<p style="font-size:17px; background-color:#00dd77;">22.10.2010 Transduction with old mVenus Stocks for taking pictures</p>===
 <br />
 <b>The motivation</b>
+We transduced HT1080 with our stocks packaged with CMV_mVenus.
 <br />
 <b>The plan</b><br />
@@ Line 1,833: / Line 2,151: @@
 <th width="100">24.9</th>
 <th width="100">25.9</th>
-<th width="100">26.9</th>
+</tr>
-<th width="100">27.9</th>
+<tr>
-<th width="100">28.9</th>
+<td>Action</td>
-<th width="100">29.9</th>
+<td>seeding HT1080</td>
-<th width="100">30.9</th>
+<td>Transduction</td>
+<td>-</td>
+<td>Fluorescence microscopy</td>
+</tr>
+</table>
+<b>The results</b>
+<gallery widths=300px heights=300px perrow=2 caption="Transduction with vectors pSB1C3_001_RC_IRCK_P5tataless as R/C and CMV_mVenus">
+Image:Freiburg10_transduction_CMV_mVenus_1.png|
+Image:Freiburg10_transduction_CMV_mVenus_2.png|
+Image:Freiburg10_transduction_CMV_mVenus_4.png|
+</gallery>
+===<p style="font-size:17px; background-color:#00dd77;">23.10 Testing the bystander effect of the cytosine deaminase</p>===
+<br />
+<b>The motivation</b><br />
+<br />
+The activated form of 5-fluorocytosine has the ability to diffuse through the plasma membrane. In this way cells get killed which were not transduced by the viral particles. We use an old yfp stock t determine if the transgene expression is on and to harvest the cells which are transduced with cytosine deaminase loaded AAV2.<br />
+<br /><b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">23.10</th>
+<th width="100">24.10</th>
+<th width="100">25.10</th>
+<th width="100">26.10</th>
+<th width="100">27.10</th>
+<th width="100">28.10</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>Transduce HT1080 and seed HT1080</td>
+<td>Transfer the transduced HT1080 onto the non transduced HT1080</td>
+<td>-</td>
+<td>-</td>
+<td>-</td>
+<td>Measure living cells via Neubauer cellchamber</td>
+</tr>
+</table>
+<br />
+<br />
+<b>Gene of interest: CMV_Cytosindeaminase</b>
+<ul>
+<li>P712</li>
+</ul>
+<b>Gene of interest: CMV_mVenus</b>
+<ul>
+<li>P468</li>
+</ul>
+<br />All dishes had this order:<br />
+<br />
+<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="80"> </th>
+<th width="80">1</th>
+<th width="80">2</th>
+<th width="80">3</th>
+</tr>
+<tr>
+<td>A</td>
+<td>Medium with prodrug</td>
+<td>Medium with prodrug</td>
+<td>Medium with prodrug</td>
+</tr>
+<tr>
+<td>B</td>
+<td>Medium</td>
+<td>Medium</td>
+<td>Medium</td>
+</tr>
+</table>
+<br />The concentration of the prodrug 5-fluorocytosine is 54 mmolar<br />
+<br />Cells were seeded following this scheme:<br />
+<b><font size="3">plate 1</font></b>
+<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="80"> </th>
+<th width="80">1</th>
+<th width="80">2</th>
+<th width="80">3</th>
+</tr>
+<tr>
+<td>A</td>
+<td>1*10<sup>5</sup> HT1080 transduced with CMV_Cytosindeaminase</td>
+<td>1*10<sup>5</sup> HT1080 transduced with CMV_Cytosindeaminase</td>
+<td>1*10<sup>5</sup> HT1080 transduced with CMV_Cytosindeaminase</td>
+</tr>
+<tr>
+<td>B</td>
+<td>1*10<sup>5</sup> HT1080 transduced with CMV_Cytosindeaminase</td>
+<td>1*10<sup>5</sup> HT1080 transduced with CMV_Cytosindeaminase</td>
+<td>1*10<sup>5</sup> HT1080 transduced with CMV_Cytosindeaminase</td>
+</tr>
+</table>
+<br />
+<br />
+<b><font size="3">plate 2</font></b>
+<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="80"> </th>
+<th width="80">1</th>
+<th width="80">2</th>
+<th width="80">3</th>
+</tr>
+<tr>
+<td>A</td>
+<td>2*10<sup>5</sup> HT1080 non transduced</td>
+<td>2*10<sup>5</sup> HT1080 non transduced</td>
+<td>2*10<sup>5</sup> HT1080 non transduced</td>
+</tr>
+<tr>
+<td>B</td>
+<td>2*10<sup>5</sup> HT1080 non transduced</td>
+<td>2*10<sup>5</sup> HT1080 non transduced</td>
+<td>2*10<sup>5</sup> HT1080 non transduced</td>
+</tr>
+</table>
+<br />
+The next plates were seeded with non transduced cells and 1*10<sup>5</sup> of the cytosin deaminase transduced cells
+<br />
+<b><font size="3">plate 3</font></b>
+<table border="3" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="80"> </th>
+<th width="80">1</th>
+<th width="80">2</th>
+<th width="80">3</th>
+</tr>
+<tr>
+<td>A</td>
+<td>2*10<sup>5</sup> HT1080</td>
+<td>2*10<sup>5</sup> HT1080</td>
+<td>2*10<sup>5</sup> HT1080</td>
+</tr>
+<tr>
+<td>B</td>
+<td>2*10<sup>5</sup> HT1080</td>
+<td>2*10<sup>5</sup> HT1080</td>
+<td>2*10<sup>5</sup> HT1080</td>
+</tr>
+</table>
+<b>The results</b>
+===<p style="font-size:17px; background-color:#00dd77;">25.10.2010 Transduction with several constructs for Flow Cytometry</p>===
+<br />
+<b>The motivation</b>
+We want to quantify via flow cytometry the Affibody superconstruct, Darpin HSPG-KO Construct and standard R/C with HSPG-KO.
+<br />
+<b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">25</th>
+<th width="100">26</th>
+<th width="100">27</th>
+</tr>
+<tr>
+<td>Action</td>
+<td>Transduction</td>
+<td>-</td>
+<td>FACS</td>
+</tr>
+</table>
+<b>The results</b>
+===<p style="font-size:17px; background-color:#00dd77;">BLANK EXPERIMENT</p>===
+<br />
+<b>The motivation</b>
+<br />
+<b>The plan</b><br />
+<table border="5" rules="all" cellpadding="5" cellspacing="1" style="font-size:13pt;text-align:center" >
+<tr>
+<th width="100">Days</th>
+<th width="100">-</th>
+<th width="100">-</th>
+<th width="100">-</th>
+<th width="100">-</th>
+<th width="100">-</th>
+<th width="100">-</th>
+<th width="100">-</th>
+<th width="100">-</th>
+<th width="100">-</th>
 </tr>
 <tr>
@@ Line 1,853: / Line 2,358: @@
 </table>
 <b>The results</b>
 <h1>Citations</h1>

Team:Freiburg Bioware/NoteBook/Labjournal/Cellculture

From 2010.igem.org

Latest revision as of 21:38, 27 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

14.8 Seeding different amounts of AAV293 for transfection

17.8 transduction with viral stocks 20 µg and 40 µg GOI-plasmid

16.8 Seeding AAV293 for transfection with new R/C

19.8 Seeding AAv293 for transfection with different HBS buffers

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

30.8 Seeding AAV293 for Transfection of 10 cm2 plates

4.9 Seeding cells for 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

15.9 Seeding AAV293 for testing our Affibody and other VP-mVenus constructs

19.9 Seeding AAV293 for checking two different Rep/Cap Constructs

22.9 Seeding AAV293 for transfection with different mVenus constructs for checking specific promoter activity

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

29.9 Checking location of VP proteins via fluorescence microscopy and checking fractions of centrifugation steps via fluorescence spectrometer

30.9 Production of new Standard mVenus Vector

30.9 Seeding AAV293 for production of standard TKGMK vector

30.9 Seeding AAV293 for production of vectors with mVenus missing beta globin

30.9 Seeding AAV293 for production of vectors with mVenus missing HGH

30.9 Seeding AAV293 for testing RC with HSPG knockout for functionality

30.9 Seeding AAV293 for testing RC WITHOUT HSPG knockout for functionality

12.10 Seeding AAV293 cells for transfection with VP-BAP

12.10 Seeding AAV293 cells for transfection with mVenus as gene of interest and pCerulean_VP1up_NLS_mVenus_VP2/3_HSPG-KO

12.10 Seeding AAV293 cells for transfection with mVenus as GOI and CFP_Middlelinker_VP2/3_HSPG-KO

17.10 Production of AAV2 with 587-His

18.10 Seeding AAV293 cells for transfection Superconstructs

18.10 Seeding AAV293 cells for transfection with Cytosindeaminase

22.10.2010 Transduction with old mVenus Stocks for taking pictures

23.10 Testing the bystander effect of the cytosine deaminase

25.10.2010 Transduction with several constructs for Flow Cytometry

BLANK EXPERIMENT

Citations

30.8 Seeding AAV293 for Transfection of 10 cm² plates