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The analysis of homologous recombination frequency

The analysis of homologous recombination frequency.

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The analysis of homologous recombination frequency

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  1. The analysis of homologous recombination frequency 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. DT40 clones carrying SCneo or DR-GFP in the OVALBUMIN locus are stored in liquid N2. 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). Data from the DR-GFP assay vary substantially in individual experiments. You should not use the cells that are incubated in 106/ml density, because cells’ viability may be compromised. 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. You should note that electroporation condition is different between stable transfection (550V/ 25F, BioRad) and transient transfection (250V/960F, BioRad). On the following pages, Dr. Sonoda described data of DR-GFP HR assay.

  2. DR-GFP DSB repair substrate in OVA locus knock-in construct:PuroR (#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 Representative data 5x106 cells in 0.5ml medium 1X106 cells in 100ul/Solution T + + 20ug of I-SCEI 3-5ug of I-SCEI %GFP positive cells Pulse with 250V/960uF Pulse with A30 (or B23) Plate the cells into 15ml medium Hours after transfection GFP positive cells reach maximum number at 36-48h after TF Count GFP positive cells by FACS

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

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

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