The analysis of homologous recombination frequency 1.There are two assays (artificial substrates) for double-strand break (DSB) induced homologous recombination (HR), SCneo and DR-GFP. Difference in the frequency of HR between wild-type and HR deficient clones tends to be larger in SCneo than in DR-GFP. 2.DT40 clones carrying SCneo or DR-GFP in the OVALBUMIN locus are stored in liquid N 2. 3.A HR assay using DR-GFP takes only two days, whilst A HR assay using SCneo takes more than a week (time for neo selection). 4.Data from the DR-GFP assay vary substantially in individual experiments. You should not use the cells that are incubated in 10 6 /ml density, because cells’ viability may be compromised. 5.You should use BioRad machine but not AMAXA for electoro-poration, because AMAXA gives only 50% increase in the efficiency of transient transfection whereas costs 10 folds, in comparison with BioRad. 6.You should note that electroporation condition is different between stable transfection (550V/ 25  F, BioRad) and transient transfection (250V/960  F, BioRad). 7.On the following pages, Dr. Sonoda described data of DR-GFP HR assay.

DR-GFP DSB repair substrate in OVA locus knock-in construct:Puro R (#18-922) transfection into DT40 cells Selection with puromycine Isolation of targeted clones (Wild-type; #2271) DR-GFP assay 1. By GenePulser 2. By Amaxa 5x10 6 cells in 0.5ml medium 20ug of I-SCEI Pulse with 250V/960uF + 1X10 6 cells in 100ul/Solution T 3-5ug of I-SCEI Pulse with A30 (or B23) Plate the cells into 15ml medium + %GFP positive cells Hours after transfection GFP positive cells reach maximum number at 36-48h after TF Count GFP positive cells by FACS Representative data

DR-GFP in OVA targeting vector (#18-922) made by Takenaka Cells with DR-GFP #2271: wild-type (puro R )

Amaxa; I-SCEI (#13-217) 3-5ug/SolutionT/A30 program %GFP positive cells Amaxa (I-SceI, 3ug/SolT/B23) Gene Pulser (I-SceI, 20ug/250V/975uF) done by Wang Cost Amaxa: 2000yen/TF GenePulser: 200yen/TF