October
15/10/2010
- organize 600 Million HEK293T cells
- prepare 2 liter of media
17/10/2010
- plate 100x 15cm dishes with each 5.5*10^6 cells
18/10/2010
- due to contamination 70 new plates are seeded in the afternoon
19/10/2010
- triple transfection with PEI using standard protocol for triple transfection:
- set-up of the plate:
- 1) off target: construct M23 with perfect binding site for miR122, AAV8 serotype (AAV8), Adenohelper virus 5 (Ad5)
- 2) control: pBSU6Sv40Luc2 transgene in ITRs transfected together with shuffeled capsid of the 50 clones which was capable of transducing Huh7 and HepG2 cells, Ad 5
- 3) control: pBSU6sv40Luc 2 transgene, AAV8, Ad 5
- 4) shRNA miR expression vector for miRhaat (pBSU6H1haat), AAV8, Ad 5
- 5) the same as 4)
- 6) tuning construct: perfect binding site for shRNA miRhaat (M1), AAV8, Ad5
- 7) tuning construct: imperfect binding site randomized from nucleotide 9-12 (M9), AAV8, Ad5
21/10/2010
- harvest 70 plates of Hek cells
- centrifuge: 10 min at 1500 rpm
- wash with 30ml 1xPBS
- centrifuge: 10 min at 1500 rpm
- start freeze and thaw cycles
- pour the gradient
- centrifuge in the ultracentrifuge for 2h at 10°C and 50K
- soak out the purified virus out of the 40% phase
22/10/2010
- double check the titre of capsids via dot plat and the packaged DNA via viral DNA extraction and running on an agarose gel using a virus with a known titre as a control
- mice were injected the following:
- 1) SV40-Luc2-miR122 perfect binding site in AAV8 capsid
- 2) SV40-Luc2 in our shuffeled capsid
- 3) SV40-Luc2 in AAV8 capsid
- 4) SV40-Luc2-miRhaat perfect binding site in AAV8 capsid
- 5) SV40-Luc2-miRhaat imperfect binding site (9-12) in AAV8 capsid
- 6) Sv40-Luc2-miRhaat perfect binding site double injected with H1-shhaat both in AAV8 capsid
- 7) Sv40-Luc2-miRhaat imperfect binding site double injected with H1-shhaat both in AAV8 capsid
25/10/2010
- plate 40 15 cm dishes with 5.5*10^6 cells per dish
26/10/2010
- transfect all 40 plates using PEI transfection buffer and the following set-up
27/10/2010
- bioluminescence measurements on mice from the first injection round
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