Team:Heidelberg/Notebook/Mouse Infection/October

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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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