M12-Tet-Off pTRE2pur FLAG mTRAF3細胞株の樹立

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  • Establishment of an inducible M12-Tet-Off pTRE2pur FLAG mTRAF3 cell
  • M12 Tet Off pTRE2pur FLAG mTRAF3 サイボウカブ ノ ジュリツ

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IL-4 induces IgE class switching in B cells together with CD40 stimulation. Studies on the signal transduction mechanism downstream of IL-4 receptor have revealed that NF-κB as well as Jak-STAT pathway plays an important role. We showed the possibility that alternative pathway of NF-κB was activated after IL-4 stimulation of murine B cell line M12. Alternative pathway of NF-κB is strictly regulated by constant degradation of NIK, an essential molecule for NF-κB activation. TRAF3 is the key molecule to keep the alternative pathway in an inactive state. TRAF3 is also regulated by the constant degradation by ubiquitin-proteasome system. To elucidate the mechanism of the TRAF3 degradation, we prepared Tet-Off gene expression system in M12 to inducibly express TRAF3 which was toxic when overexpressed in cells. Firstly, pTet-Off vector was introduced into M12 cells by electroporation and G418 resistant clones were selected which harbored pTet-Off. Clones were further selected to show high responsiveness to Doxycyclin withdrawal using transient expression of luciferase vector. One of the clones, C'' , was transfected with pTRE2pur-mTRAF3 vector to obtain FLAG-tagged mTRAF3 expressing cells upon induction. One clone, C'' -1' , was successfully established which expressed mTRAF3 in an inducible manner.

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