16.8 Seeding AAV293 for transfection with new R/C

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

The plan

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

The transfection was performed according to standard protocol.

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

The results

As you can see the eGFP constructs worked very well. And even the transduction efficiency of the standard R/C is doubled, the conclusion is that in previous transfections the AAV293 cells were to confluent. The confluence of the cells is critical!

20.8

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


The Facs data at 22.8

19.8 Seeding AAv293 for transfection with different HBS buffers

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

The Plan

We try 5 different 2xHBS buffers:

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

Used Plasmids: Standard protocol: 10µg of each plasmid

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

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

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

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

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

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

Checking transduction efficiency via fluorescence microscopy

The motivation

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

The plan

1. plate 1:
• stock 1 (with 100.000 cells)
• stock 2 (with 200.000 cells)
2. plate 2:
• stock 3 (with 400.000 cells)
• stock 4 (with 500.000 cells)
3. plate 3:
• stock 5 (with 800.000 cells)
• stock 6 (with 1.000.000 cells)
4. plate 4:
• stock 7 (with 1.200.000 cells)
• stock 8 (with 1.500.000 cells)
5. plate 5:
• stock 9 (with 1.750.000 cells)
• stock 10 (with 1.750.000 cells)

The results

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

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

The motivation

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

The plan

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


We dont really know the reason for the quite low average YFP expression, but the remarkable note is, that the construct (pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR) fusioned from the created Biobricks still works!

30.8 Seeding AAV293 for 10xTransfection

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

The plan

Transfection will be done with:

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

0,5 ml of each stock become deepfreeze in.

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

XXXXXXX getting valide data of 10µg samples

The motivation
we want to check the YFP expression
The plan

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

the results

28.8 Seeding AAV293 for checking functionality of RC vector

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

The plan

Transfection was done with:

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

The results

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

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

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

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

The plan

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

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

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!

19.9 Seed AAV293 for testing pTERT promoter

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.

20.9 Seeding AAV293 for creating first VP-mVenus-fusion constructs

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.

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. The main

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

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:

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
Interpretation and discussion of our results.

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 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
Interpretation and discussion of our results.

30.9 Seeding AAV293 for testing RC WITHOUT HSPG knockout for functionality

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.

Experiment

The motivation
The plan

The results

Citations

[1] Media:Freiburg10_Stratagene_AAV_helper_free_manual.pdf
[2] Titel einfügen
[3] Read, A. P.; Strachan, T. (1999). "Chapter 18: Cancer Genetics". Human molecular genetics 2. New York: Wiley. ISBN 0-471-33061-2.
[4] Titel einfügen
[5] Media:Freiburg10 Titration of AAV-2 particles via a novel capsid ELISA.pdf