Figure 1. Scheme of the DTG system. A. Scheme of tetR and TA tagged KR expression in the U2OS TRE cell line. To induce ROS-mediated damage at a specific locus in the genome, we fused KR to the tetracycline repressor to induce ROS damage in a 90 kb tetracycline response element (TRE) array (totally 96 repeats) in U2OS cells. B. Expression of tetR-KR in U2OS TRE cell line.

Materials and Reagents

1. U2OS-SCE cell line (made in our laboratory) (Lan et al., 2010)
   Notes:
   1. In this cell line, 200 copies of pTRE / I-SceI were integrated in U2OS cells.
      
   2. Cells are cultured with Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (EuroClone S.p.A. P.IVA, catalog number: ECM0103L ) with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F9665 ).
      
   3. For preparation of cells, cells were washed with PBS without Ca²⁺ and Mg²⁺ (EuroClone S.p.A. P.IVA, catalog number: ECB4004L ), trypsinized with Trypsin/EDTA without out Ca²⁺ and Mg²⁺ (Thermo Fisher Scientific, catalog number: BW17161E ).
2. Plasmids
   
   1. pBROAD3/tetR-KR
      
   2. pBROAD3/tetR-mCherry
      
   3. pBROAD3/TA-KR 
   4. pBROAD3/TA-mCherry
      Note: pBROAD3/tetR-mcherry was provided by Dr. Edith Heard (Masui et al., 2011); pBROAD3/tetR-KR, pBROAD3/TA-mCherry, pBROAD3/TA-KR were made in our laboratory (Lan et al., 2014). Sequences are available based on requests.
      
3. GFP-fusion protein or endogenous protein
   
4. Lipofectamine 2000 (Life Technologies, catalog number: 12566-014 )
   
5. Opti-MEM (Life Technologies, catalog number: 51985-091 )
   
6. 35 mm glass bottom culture dishes (MatTek, catalog number: P35GC-1.5-14-C )
   
7. IMMOIL-F30CC (Chip Humphries, catalog number: Z-81225 )

Equipment

1. 35 mm glass bottom culture dishes
   
2. 37 °C, 5% CO₂ cell culture incubator
   
3. Olympus FV1000 confocal microscopy system (OLYMPUS, model: FV1000- FILTER DETECT ; SYS.PACKAGE: IX81-1 405/M_AR/559/635N) with 488, 559 nm lasers
   
4. PLAPON 60x oil lens (super chromatic abe. corr. obj W/1.4NA FV) (OLYMPUS, catalog number: FM1-U2B990 )
   
5. Thermo-plate (MATS-U52RA26 for IX81/71/51/70/50, metal insert, HQ control) (OLYMPUS, catalog number: OTH-I0126 )
   
6. 15 watt cool white fluorescent bulb (OSRAM SYLVANIA)
   
7. A stage UVP (Upland, CA)
   

Procedure