title: C2C12 cells expressed GFP-H3.3 at undifferentiated state
organism: Mus musculus
description: 0.5% formaldehyde 5min fixationThe H3f3a (purchased from Operon Biotechnologies) used for expression of H3.3. The cDNA were ligated into the Bidirectional Tet Expression Vector pT2A-TRETIBI (modified Clontech Tet-On system), which contains TolII transposon elements and EGFP cDNA located upstream of the cDNA sequence and which was modified from pT2AL200R150G (provided by Dr. Kawakami). The pT2A-TRETIBI/EGFP-H3.3 transfection was performed using Lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA). C2C12 cells at 20% to 30% confluence were transfected with an expression vector, pCAGGS-TP coding transposase (provided by Dr. Kawakami), and pT2A-CAG-rtTA2S-M2 and incubated for 24 h. To create cell lines stably expressing GFP-H3.1 , transfected cells were cultured for 14-21 days in the presence of doxycycline and G418. Finally, the GFP positive cells in the stable lines were selected using fluorescence activating cell-sorting.