T-REx-293 cells (Invitrogen), which constitutively expresses the Tet repressor (TetR), were transfected with pcDNA4/TO-coF14-TAP plasmid, which encodes F14 under the control of the HCMV immediate early promoter and two tetracycline operator (TetO2) sites. Transfected cells were selected in the presence of blasticidin (10 μg ml-1) and zeocin (100 μg ml-1) and clonal cell lines were obtained by limiting dilution. Expression of protein F14 within these clones was analysed by immunoblotting and flow cytometry with anti-FLAG antibodies. T-REx-293-EV, T-REx-293-B14 and T-REx-293-C6 cell lines were described elsewhere21 (link).
Protocol detail
Inducible Expression of Protein F14
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Anti antibodies
Cell lines
Cells
Clones
Dilution
Flow cytometry
Hcmv
Plasmid
Protein
T cell
Tet repressor
Tetracycline
Zeocin
Corresponding Organization : University of Cambridge
Other organizations : Wellcome/Cancer Research UK Gurdon Institute
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Variable analysis
independent variables
- Transfection of pcDNA4/TO-coF14-TAP plasmid encoding F14 under the control of the HCMV immediate early promoter and two tetracycline operator (TetO2) sites
- Expression of protein F14 within the clonal cell lines, analyzed by immunoblotting and flow cytometry with anti-FLAG antibodies
- T-REx-293 cells (Invitrogen), which constitutively expresses the Tet repressor (TetR)
- Selection of transfected cells in the presence of blasticidin (10 μg ml-1) and zeocin (100 μg ml-1)
- Clonal cell lines obtained by limiting dilution
- T-REx-293-EV, T-REx-293-B14 and T-REx-293-C6 cell lines described elsewhere
- Not explicitly mentioned
- Not explicitly mentioned
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