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

• 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

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

Plate 2 YFP: 150.000 cells per well

Plate 3 YFP: 200.000 cells per well

• (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

The following amount of cells were seeded in 10 cm²:

1. 100.000
2. 100.000
3. 200.000
4. 200.000
5. 300.000
6. 300.000
7. 400.000
8. 400.000
9. 500.000
10. 500.000
11. 600.000
12. 600.000
13. 700.000
14. 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

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

• 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

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.

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

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

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:

1. DMEM as negative control
2. resuspended Pellet with mVenus-fusioned viral capsids
3. resuspended Pellet with "standard virus"
4. supernatant with mVenus-fusioned viral capsids
5. 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.

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.

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

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.

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.

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.

The results

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.

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

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

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

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.

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

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

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
The plan

The results

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.

The plan

Gene of interest: CMV_Cytosindeaminase

• P712

Gene of interest: CMV_mVenus

• P468

All dishes had this order:

The concentration of the prodrug 5-fluorocytosine is 54 mmolar

Cells were seeded following this scheme:

The next plates were seeded with non transduced cells and 1*10⁴ of the transduced cells

The results

BLANK EXPERIMENT

The motivation
The plan

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