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Contents 1 146. labday 11.10.2010 1.1 Colony PCR of Cloning VP2 Fusion and Super constructs into pSB1C3 1.2 Preparation of SDS-PAGE gel (10%) 1.3 Colony PCR of p40_VP123_capins 1.4 Cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD 1.5 Seeding HT1080 and A431 for testing different concentrations of ganciclovir by MTT-Assay 1.6 FACS-Analysis 1.7 Preparation of the ELISA 1.8 Trafo evaluation of Rep52 2 147. labday 12.10.2010 2.1 Sequencing analysis: pSB1C3_mGMK_TK30 and pSB1C3_CD 2.2 Test digestion of pSB1C3_CD and pSB1C3_CD_SDM-PstI_new 2.3 qPCR of virus stock: P816/ TKGMk-Standard; R/C: P468 from 5.10. (VLP harvested by Adrian) 2.4 Cloning sr39 into mGMK_TK30 2.5 Concentration of virus stock for Western Blot analysis 2.6 Transduction of HT1080 and A431 2.7 Mini-Prep and test digestion of several constructs 2.8 Repetiton: Site-directed mutagenesis in pSB1C3_CD construct 3 148. labday 13.10.2010 3.1 qPCRs of virus stocks 3.2 SDS Page of unmodified AAV2 capsids 3.3 Midi-Prep 3.4 Repetiton: Test digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_SDM-PstI_hgH_rITR 3.5 Sequencing analysis: pSB1C3_leftITR_pCMV_betaglobin_mGMK_TK30_hgH_rITR (P825) 3.6 Repetition of cloning of Rep into Rep78 3.7 Midi-Prep 4 149. labday 14.10.2010 4.1 Continuation of Western Blot of unmodified AAV2 capsids 4.2 Cloning hGH_rITR into (form P728) into pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_SDM-PstI (P822) 4.3 Cloning of the ViralBricks 453-empty and 587 empty 4.4 SDS PAGE of viral capsids 4.5 Sequence analysis of super constructs 4.6 Transfection with pCMV, p40, pAAV_RC 4.7 Trafo evaluation of Rep78 4.8 Picking clones 5 150. labday 15.10.2010 5.1 Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729) 5.2 Midi-Prep 6 151. labday 16.10.2010 7 152. labday 17.10.2010 8 153. labday 18.10.2010 9 154. labday 19.10.2010 10 155. labday 20.10.2010

146. labday 11.10.2010

Colony PCR of Cloning VP2 Fusion and Super constructs into pSB1C3

Investigator: Achim, Hanna

Comment: Because yesterday's cloning didn't deliver a good separartion of the expected gel bands. Nevertheless ligation and trafo was performed. In order to immediately find out, whether we received successful results a colony PCR will be performed.
Two clones were picked from each plat. In addition to that a positive (pAAV_RC) and a negative control (pSB1C3_lITR) was prepared.
Used primer: 4200 rev and Cap3500 for. Expected fragment size: 885 bp.
PCR was performed following the standard protocol.

All samples match the positive control!
To do: Mini-Prep and sequencing.

Preparation of SDS-PAGE gel (10%)

Investigator: Hanna

Comment: In order to perform a Western Blot of different virus capsids (with and without capsid-motifs), 2 10% SDS polyacrylamid gels were prepared.

Resolving gel: 15 mL

• H₂O: 5.9 mL
• Acryl-bisacrylamide mix (30%): 5 mL
• Tris (1.5 M, pH 8.8): 3.8 mL
• SDS (10%): 0.15 mL
• Ammonium persulfate (10%): 0.15 mL
• TEMED: 0.006 mL

Stacking gel (5%): 5 mL

• H₂O: 3.4 mL
• Acryl-bisacrylamide mix (30%): 0.83 mL
• Tris (1.5 M, pH 6.8): 0.63 mL
• SDS (10%): 0.05 mL
• Ammonium persulfate (10%): 0.05 mL
• TEMED: 0.005 mL

Colony PCR of p40_VP123_capins

Investigator: Bea

Comment: Since the first attempt did not work,and no cells grew on the plate and the same ligation was transformed again into BL-21 and a lot of clones grew on the plate, I decided to perform a colony PCR in order to check several colonies and to inoculate at the same day for a Midi-Prep.

Protocol:

• Primer used: O162
• Primer used: O38

The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture:

Result: We can see two things: The cloning of p40 to VP123 worked quiet well AND the Robust PCR Kit which was used for the first time worked as well.

Cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD

Investigator: Stefan

Comment: To produce another GOI for testing in cell culture, the cytosine deaminase needs to be assembled with lITR_promotor_betaglobin. In the next step hgH_rITR needs to be added.

Vector name:
pSB1C3_CD clone 1
pSB1C3_CD clone 2

Insert name:
pSB1C3_lITR_CMV_beta-globin (P729)
pSB1C3_lITR_phTERT_beta-globin (P730)

Digestion:

components	volume CD clone 1 + 2 /µl	volume P729 /µl	volume P730 /µl
DNA	6	14	6
BSA (10x)	2	2	2
Buffer 4 (10x)	2	2	2
Enzyme EcoI	1	1	1
Enzyme XbaI	1	-	-
Enzyme SpeI	-	1	1
H2O	8	-	8
Total volume (e.g. 15,20,25,30 µl)	20	20	20

Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt

CD clone 1 yielded to bands around 2500 bp to 3000 bp. Since the vector was cut only using EcoRI and SpeI, it was expected to be linearized, not to be cut into two fragments this size. Therefore, this sample was discarded and cloning was continued using CD clone 2.

Gel extraction:
Was performed according to protocol.

T4 Ligation:

ligation name	729 + CD cl2	730 + CD cl2
volume of vector	3,67	2,82
volume of insert	4,33	5,18
T4 ligase buffer (10x)	1	1
T4 ligase	1	1

Transformation:
Was performed according to standard protocol using BL21 cells.

Seeding HT1080 and A431 for testing different concentrations of ganciclovir by MTT-Assay

Investigator: Kerstin, Anissa

• Seeding 4x 96-well plates: 2x HT1080 and 2x A431

FACS-Analysis

Investigator: Kerstin

...

Preparation of the ELISA

Investigator: Volker

The AAV particle standard that contains 3.6x10^9 viral particles was dissolved in 500µl as described in the protocol of the Progen AAV Titration ELISA and a absorption spectrum was measured. These purified and concentrated viral particles could be used for biophysical measurements, there for the possibility to detect the viral particles by absorption was interesting for us.

The Spectrum measured in the NanoDrop is the following:

Trafo evaluation of Rep52

Investigator:Kira
T4 ligation seems to worked out. The plate contained many colonies.

147. labday 12.10.2010

Sequencing analysis: pSB1C3_mGMK_TK30 and pSB1C3_CD

Investigator: Stefan

Test digestion of pSB1C3_CD and pSB1C3_CD_SDM-PstI_new

Investigator: Stefan

Comment: Nachricht von Stefan an Stefan: bitte ergänzen!

Comment: Sequencing results revealed that there is CFP rather than CD in P819. In order to verify this and exclude the possibility that the wrong plasmid was sent for sequencing test digestions were performed. Additionally the original vector at which the SDM to remove the PstI was performed was digested as well to verify its insert.

Components	digest 1 / µl	digest 2 / µl
DNA	1	7
Buffer 4	1	1
BSA (10x)	1	1
Enzyme I	SpeI 0,3	PstI 0,3
Enzyme II	XbaI 0,3	XbaI 0,3
H2O	6,4	0,4
Total volume	10	10

Comment: Using these digestion approaches, it was expected for CFP to deliver always bands at ~750 bp. For the CD it was expected to show bands at 90 bp, 1200 bp and 2000 bp if the CD still contains the PstI restictiton site, 1300 bp and 2000 bp if it was removed.

Gel:
0,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes

Comment: As it can be seen, P819 does contain CFP, therefore a mistakenly sequenced plasmid can be excluded. Also it can be seen that P690 contains the CD. Therefore an additonal SDM of P690 to remove the PstI restriction site is necessary.

qPCR of virus stock: P816/ TKGMk-Standard; R/C: P468 from 5.10. (VLP harvested by Adrian)

Investigator: Achim

Cloning sr39 into mGMK_TK30

Investigator: Bea

Comment: We do not only want to submit the tk30, a mutant version of the thymidine kinase, but as well another mutant protein which is named sr39. SR39 seems to have a better activity than the tk30 without being fused to the guanylate kinase (mgmk), but the sr39 also works quite well as a fusion protein. We ordered one part of the sr39 and we are now subcloning it into the mgmnk_tk30 construct in order to obtain mgmk_sr39.

Protocol:

The digested fragments were loaded on a 1.2% agarose gel. The results can be seen above in the gel picture: I cut out the small visible band in the right lane, and the upper intensive band of the left lane.

Result:

After gel extraction has been performed, I ligated the two fragments and transformed BL-21 cells, plated them on cm agar plates and incubated them over night at 37°C.

Concentration of virus stock for Western Blot analysis

Investigator: Hanna

For the first Western Blot try, we investigate whether is is enough to concentrate the virus stock (from cell supernatant) and then to simply load it onto a SDS-PAGE gel and perfom immuno-staining.
For this purpose 8 mL virus stock (pHelper, pAAV_RC, GOI=YFP) were concentrated via amicon filtration.

• First, amicon filter was washed with 4 mL PBS.
• Then 4 mL virus stock was loaded onto the filter and centrifuged at 3000 rpm for 15 minutes.
• Flow-through was discarded and additional virus stock was loaded, centrifuged at 3000 rpm for 25 minutes.
• Flow-through was discarded, remaining solution was re-suspended and additional virus stock was added. Amicon filter was centrifuged at 3000 rpm for 50 minutes.
• Flow-through was discarded. Residual protein solution: 500 µL. Filter was washed with 3 mL PBS, centrifuging 35 minutes two times.
• 2 x 40 µL protein solution (16x) was taken and mixed with Laemmli buffer and stored over night @ 4°C.

To do: SDS-PAGE and Western Blot tomorrow.
Perfomance of another try by harvesting viruses of the cell pellet with RIPA buffer & freeze/ thaw.
Western Blot of unmodified, and modified capsids (N-terminal fusion and VP1 insertion with CFP ref. mVenus) --> size shift should be detectable.

Transduction of HT1080 and A431

Investigator: Kerstin

Mini-Prep and test digestion of several constructs

Investigator: Stefan

Glycerol stocks were prepared:

• B651 = pSB1C3_P40_VP123
• B652 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_PstI_SDM_hgH_rITR
• B653 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_PstI_SDM_hgH_rITR
• B654 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR
• B655 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR
• B656 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO
• B657 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO
• B658 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO
• B659 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO
• B660 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR
• B661 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR

Mini-Prep was performed according to standard protocol:

• P821 = pSB1C3_P40_VP123 c = 327,4 ng/µl
• P822 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 342,9 ng/µl
• P823 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 324,0 ng/µl
• P824 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 81,6 ng/µl
• P825 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 148,4 ng/µl
• P826 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 105,8 ng/µl
• P827 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 121,1 ng/µl
• P828 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 188,4 ng/µl
• P829 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 140,4 ng/µl
• P830 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR c = 197,2 ng/µl
• P831 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR c = 207,3 ng/µl

Test digestion:

Components	P822 + P823 / µl	P824 + P825 / µl	P830 + P831 / µl
DNA	1,5	1,5	2
Buffer 4	1	1	1
BSA (10x)	1	1	1
PstI	0,3	-	-
XbaI	0,3	0,3	0,3
SpeI	-	0,3	0,3
H2O	5,9	5,9	5,4
Total volume	10	10	10

Gel:
0,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes

Comment: P822 and P823 smear a lot, therefore evaluation is difficult. Another test digestion would be reasonable.
P824 contains hgH_rITR, therefore it will be discarded. P825 looks well and was sent for sequencing.
P830 and P831 look well and will be sent for sequencing tomorrow.

Repetiton: Site-directed mutagenesis in pSB1C3_CD construct

Investigator: Stefan

Comment: Sequencing revealed that there is no CD in our pSB1C3_CD but rather CFP. So, anonther approach of the SDM needs to be performed.

Protocol using the QuickChange Lightning Kit from Stratagene:

Ingredients:

Ingredients	Volume / µl
10x reaction buffer	2,5
dNTP	0,5
forward primer: O191	0,56
reverse primer: O192	0,57
DNA Template	0,5 (1:10 dilution)
QuikSolution Reagent	0,75
QuikChangeLightning Enzyme	0,5
H2O	14,35
Total volume	25

The following PCR program was used:

• 95 °C 120 s
• 95 °C 20 s
• 60 °C 60 s
• 68 °C 105 s
• Goto step 2 repeat 17 times
• 68 °C 300 s

Digestion using DpnI:
To digest methylated and hemi-methylated DNA, 1 µl of DpnI was added and the mixture was incubated for 10 minutes at 37 °C.
Transformation:
Trafo was performed according to protocol, but using XL1b cells instead of XL-10 Gold cells.

148. labday 13.10.2010

qPCRs of virus stocks

Investigator: Achim

Results:

SDS Page of unmodified AAV2 capsids

Investigator: Hanna

Comment: Yesterday a 8x concentration of AAV2 supernatant-stock was performed. 10 µL Laemmli-buffer was added to 40 µL AAV stock (8x) and stored over night at 4°C.
Today a SDS-PAGE of these samples will be performed.

Samples were incubated for 10 minutes at 95°C for denaturation.

Loading plan:

5 µL PageRuler Prestained Protein Ladder

10 µL Sample

15 µL Sample

25 µL Sample

After running front reached the end of the gel, PAGE was stopped.

Western Blot:
Three layers of transfer buffer soaked whatman paper were placed onto the blotter. A polyvinylidene difluoride membrane was activated by methanol (5 seconds) and then incubated for 5 minutes in transfer buffer. Membrane was then placed onto the whatman paper, gel was carefully layed on top. Air bubbles were removed, ensuring elictricity flow. After coverage by 3 further layers of whatman, blotting was performed at 200 mA for 1 hour.

Blocking:
In order to minimize nonspecific antibody interactions, membrane was incubated in blocking buffer (TBS (pH 7.5), 5% milk powder) for one hour, shaking.

Primary antibody incubation:
A 1:10 dilution of the B1 antibody in TBS + 1% milk was prepared. Membrane was incubated in 50 mL falcon over night at 4°C, rotating.

Midi-Prep

Investigator: Chris W.

Midi-Prep of:

pSB1C3_CMV_VP123_453_Z34C =P832 =B656
pSB1C3_CMV_VP123_453_RGD_HSPG-KO =P833 =B658
pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 1 =P834 =B561
pSB1C3_001_RC_IRCK_P5tataless clone 1 =P835 =B516
pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P836 =B200
pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P837 =B200

The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no.	P832	P833	P834	P835	P836	P837
concentration (ng/µl)	3615,8	1504,7	x	x	x	x

Comment: P834-837 given to sven

Repetiton: Test digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_SDM-PstI_hgH_rITR

Investigator: Stefan

Comment: Repetiton of yesterday's test digestion which yielded no clear band at 2000 bp as it was expected.

Components	P822/P823 / µl
DNA	1,5
Buffer 4	1
BSA (10x)	1
SpeI	0,3
XbaI	0,3
H2O	5,9
Total volume	10

Gel:
0,45g agarose, 50 ml TAE (0,9%), 3 µl GELRED, 90 Volt, running time ~110 minutes

Comment: This time both samples look well. P822 will be sent for sequencing.

Sequencing analysis: pSB1C3_leftITR_pCMV_betaglobin_mGMK_TK30_hgH_rITR (P825)

Investigator: Stefan

For sequencing the following primers were used_

• qPCR primer CMV for (O20)
• GOI primer for (O30)
• seq. primer TK GMK for (O23)
• TK30 SDM Pst for (O191)

Sequencing comfirmed that the pysical sequence fits our theoretical. Next step will be to remove the remaining PstI restriction site within TK30.

Repetition of cloning of Rep into Rep78

Investigator: Kira
Comment: The last 2 approaches to digest Rep78 and to clone failed because Rep78 was almost gone after PCR purification and was not detectable on the gel, even if I used the double amount of DNA. Thus, new over night culture was grown in order to perform the experiment again

Regarding the activity conditions of the enzymes, digestion was performed in 2 steps.

Components	Rep78
DNA	3 µl
BSA (100x)	2 µl
Buffer no. 3	2,0 µl
Enzyme SwaI	1,0 µl
H2O	12 µl
Total volume	20

incubation @ 25 C for approx. 2 h
Dephosphorylation was performed because of the blunt cleavage. PCR phosphorylation followed in order to get rid off the buffer because Buffer 3 is not appropriate for further digestion.

Components	Rep78
DNA	30 µl
BSA (100x)	0 µl
Buffer no. 2	4,5 µl
Enzyme HindIII	1,0 µl
H2O	9,5 µl
Total volume	45

incubation for 2h at 37C

File:Rep78 2010 10 13.jpg

Ligation with T4 ligase

v(insert)=4,4 ul
v(vector)= 3,6 ul
T4 buffer= 1 ul
T4 ligase= 1 ul

incubation at RT for 45 min

Transformation according to the standard protocol with BL21

Midi-Prep

Investigator: Chris W.

Midi-Prep of:

pSB1C3_CMV_VP123_capins =P862 =B474
pSB1C3_P40_VP123 =P863 =B651

The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no.	P862	P863
concentration (ng/µl)	3310,5	2209,2