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