Team:Freiburg/Notebook/lab epigenetics

µl	Substance
1	Plasmid
1	buffer 4
0,5	EcoRV-HF
0,5	NotI-HF
7	H2O

• Incubation: 1,5h at 37° C

11 out of 12 clones were positive, as all 3 bands were visible as expected. Numer 3 and 5 were sent for sequencing with oIG0009,8007 and 8008.

µl	Substance
5	Plasmid
5	buffer 2.1
2	BbsI
39	H2O

• Incubation: 2h at 37° C

loading scheme: marker - pIG8002(digested) - pIG8002(control) - pIG8004(digested) - pIG8004(control). No big shift is visible when loading only undigested control.

digested bands were cut and extracted using the Qiagen gelEx kit. Yields were roughly 20ng/ul. The next day they will be ligated with the oligos.

µl	Substance
2,5	vector
2	insert
1	T4 buffer
0,5	T4 Ligase
4	H2O

• Incubation: 1h at 22° C

5ul of each transformation assay was transformed into competent E.coli (batch 8.5.), following the standard protocol.

4.7. Test digest of ligated crRNAs

Clones were obtained for all constructs. Minipreps were performed and yielded roughly 230ng/ul. Test digest was performed with BbsI and XhoI as described several times above. Results are inconclusive, so pIG8002_1 and pIG8004_1 were sent for sequencing with oIG0015.

5.7.

pIG8002_1 was sequenced as correct, pIG8004_1 was negative, BbsI seemed to have cut too much of the plasmid, one DR is missing. Strange. rest was sent for sequencing with oIG0015.

9.7.

Sequencing revealed positive clones for pIG8002_1 and 3, as well as for pIG8004_4. Negative colonies were discarded. More preps were done to screen for further clones.

• 8002_2.2
• 8002_4.2
• 8004_1.2
• 8004_2.2
• 8004_3.2

10.7.

8004_3.2 is positive, rest negative. For the missing constructs one of each clone was sent.

11.7.

positive clones were obtained for 8002_2.3, 8002_4.3, and 8004_1.3. New clones were sent for sequencing.

12.7.

positive clone for 8004_2.4 was found. All constructs are done. Midi preps were inoculated in 150ml LB with 150ul Amp. As well as control plasmids without target sites. Flasks were directely inoculated by giving a little bit of colony with a sterile tip into the flask.

15.7.

Midi preps were performed using the JetStar Midi Kit. Pellet was resolved in 100ul of ddH2O. All preps yielded acceptable results (500-1900 ng/ul).

HEK293T cells were seeded into 24 well plates. Our construct should be able to efficiently repress VEGF expression by methylating H3K9. As HEK293 cells have an open locus we should see a reduction in VEGF levels. 65.000 cells were seeded per well.
Each construct will be transfected in biological triplicates, let grow for 48 hours and the supernatant harvested. ELISA measurments will be performed using the peprotech VEGF ELISA kit.

16.7.

Transfection of the cells was performed as described in the table below. PIF-GFP was transfected as a transfection control to have a fast impression of transfection efficiency. Next transfection should be calculated for 2-3 wells more to have a buffer for pipetting mistakes. Recalibrate pipette?

17.7

Transfection seems to have worked, as we see green fluorescence in the nuclei of the cells.

VEGF ELISA kit arrived. Antibodies were aliquoted following the instructions and stored at -20°C. Buffers were prepared as follows:

• PBS: dilute 100ml 10xPBS in 900ml ddH2O.
• PBS-T: 1xPBS+0,05% Tween-20
• Blockbuffer: 31ml PBS+ 0,31g BSA
• Diluent: 45ml PBS-T+0,045g BSA

every buffer containing BSA is stored at 4°C. ELISA plate was coated with capture antibody as described in the instructions.

18.7. ELISA detection of VEGF content of supernatant

All steps were performed following the protocol of Peprotech, except the following steps: Blocking time was elongated to 2 hours, and washing was performed 3 times with 300ul wash buffer(PBS-T). Samples were used diluted and 1:2 diluted for ELISA detection.

The loading scheme of the ELISA plate is shown below.

results of ELISA

direct comparison of 8002 and 8004 constructs show less VEGF in 8002(active G9a) as expected. controls show very low amounts as well. This is rather strange, but gives a hint that our primary idea is correct. Several possible problems and solutions wre identified.

• VEGF is secreted when cells are exposed to oxygen stress. The distribution of O2 could be varying inside the plate, messing with our results. The use of filters instead of lids could be a solution.
• The use of an internal standard could be a possible solution for reducing cell toxicity of G9a and gives us as well a hint on cell number/transfection efficiency. A SV40:SEAP plasmid will serve as this.
• Triplicates should be mandatory, if possible

22.07.

HEK293T cells were seeded at70.000 cells/well in 24 well plates.

23.07.

transfection was performed as described in scheme and protocol from 16.07.. Each well was co-transfected with 0,0375ug of SV40:SEAP (commercial pK2 SEAP control plasmid by CloneTech (R)) that will serve as internal standard. The cells were quite dense, so we will have to evaluate transfection efficiency carefully.

24.07

Cells were too dense, transfection efficiency was low => Cells were discarded. Seeding of cells will be repeated.

27.07.

HEKs were seeded at 65.000 cellsper well in 24 well plates. We were advised by Bea to assure good distribution of cells.

28.07.

Cells show strange behaviour: They form a clump in the middle of the well, whereas the outer space is too lose. This will reduce the transfection efficiency strongly in the middle of the well, making our experiment quite difficult. Cells were discarded. again, cells were seeded with the help of Adrian Fischer.

29.07.

Same procedure as everyday. We suspect the cells to be misbehaving. We acquired new cells from AG Warscheidt for testing other cells. A contamination could explain the problems in growth.
for testing we transfected the old cells anyway to check efficiency.

New cells were seeded at 65.000 cells per well into 24 well plates. (HEK293 cells). Cells were resuspended and singled with great(!) caution. We checked the transfection efficiency of the old cells which was below 10%. The cells were discarded.

01.08.

Cells are not dense enough for transfection (~30%), so transfection was postponed to the next day.

2.8.

Cells were transfected following the standard protocol as described in the following tables at 12.a.m..

The plates were covered with O2 diffusible filters to prevent oxygen stress to occur and messing up our data.

03.08.

Medium of transfected cells was changed at 10 a.m., to repress the native VEGF level to a minimum. Transfection efficiency was around 50% when assaying the PIF-GFP construct. We are confident, that this will suffice for our experiment.

04.08

Supernatant of cells was harvested (400ul) for further analysis. 200ul are used for the VEGF ELISA, whereas the other 200ul were frozen to perform a SEAP measurement the next days as internal standard. ELISA was performed as described above.
Additionally cell lysis was performed using modified RIPA buffer for expression control of our construct by Western blot. Lysates were cooked for 5 minutes in sample buffer and frozen for later blotting.

VEGF locus 2 and 3 were displaying strong (!) repression this time (see graph) in comaprison to the control bar. But controls are varying strongly in VEGF expression. Even more, locus 1 behanves really strange. Maybe the cells have died in this wells? Western Blot will show the problems.

05.08.

SEAP measurement was performed as described in the standard protocol. All VEGF levels were divided by the SEAP level to get a normalization to an internal standard. Error was propagated using Gauß's mistake propagation.

by looking at the data now it is obvious that sample 4_1 can be discarded for further analysis. But nevertheless the rest of the samples is looking great. Even the controls are within range of one standard deviation. Strangely PIF:GFP displayed a very strong SEAP expression, lowering the bar in the normalized plot. But loci VEGF2 and VEGF3 display really strong effects.

For further expression analysis we were using a Western blot approach. To do so, 2 8% Acrylamide gels were pourn with 50% Sucrose added. They were stored at 4°C and will be ran tomorrow.

06.08.

Protein samples were run on an 8% gel at 130Volt. Afterwards a semidry blot was performed at 70mA to transfer protein onto an VDF-membrane. Primary antibody (anti-HA and anti-ß-act) was diluted 1:2500 in 2% milkpowder. Blocking was performed using 4% milk in PBS-T for 1 hour. Primary incubation took place overnight at 4°C on a vertical shaker.

07.08.

Membrane was washed 3x for 5 minutes with 1x PBS-T. Secondary antibody (anti-mouse) was diluted 1:5000 and applied to the membrane for 2 hrs at RT. An additional washing step was performed 3 times for 5 minutes in 1x PBS-T.

Detection of Western Signal<

Membranes were treated with 600ul of ECLI and II each. Signal was detected using the LS imageQuant. Results are displayed in the following figure.

We see expression of dCAS9:G9a in each well, except 4_1, which supports our hypothesis, that here a problem with the cells occurred. The ß-actin levels are varying strongly what leads to the impression, that cell lysis was not performed perfectly . Nevertheless, we could detect our protein in each well were we saw effect. Today is a good day!

08.08.

Further planning after discussion: A kinetic should be done, but should be planned carefully.

12.08.

Plans for Kinetics:

• Kinetics will be performed in two ways: Either measuring production of VEGF in 24hours over 5 days, and VEGF levels over 5 days, without medium change.
• To reduce variability in between plates we will transfect in 10cm dishes, and spread afterwards in 24 well plates.
• 6 24-well plates will be used
• Filters and SEAP standard will be used

14.08.

All constructs were transfected into HEK293 cells in 10cm-dishes. For DNA mix, see the following table. Every construct was treated the same.

15.08.

To detach cells from the plates, medium was removed. Afterwards they were washed in 5ml PBS.
PBS was removed and another 10ml of PBS was added. cells detached using a cell scraper centrifuged and resuspended in 4ml DMEM. 75% of a plate was diluted in 9.5ml medium. 500ul was put to each well of a 24 well plate. Plates were seeded according to following scheme.