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The analysis of homologous recombination frequency
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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.
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
5x106 cells in 0.5ml medium
+
20ug of I-SCEI
Pulse with 250V/960uF
2. By Amaxa
Representative data
1X106 cells in 100ul/Solution T
+
3-5ug of I-SCEI
Pulse with A30 (or B23)
Plate the cells into 15ml medium
Count GFP positive cells by FACS
%GFP positive cells
1. By GenePulser
Hours after transfection
GFP positive cells reach maximum
number at 36-48h after TF
DR-GFP in OVA targeting vector (#18-922) made by Takenaka
Cells with DR-GFP
#2271: wild-type (puroR)
Amaxa (I-SceI, 3ug/SolT/B23)
Cost
Amaxa: 2000yen/TF
GenePulser: 200yen/TF
Gene Pulser (I-SceI, 20ug/250V/975uF)
done by Wang
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%GFP positive cells
%GFP positive cells
PCNA1
PCNA2
2.5
WT
PCNA1
2
PCNA2
1.5
1
0.5
0
0
12
24
36
Amaxa; I-SCEI (#13-217) 3-5ug/SolutionT/A30 program
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60
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