1. Homology-directed gene repair
exogenous
repair substrate
I-SceI xx
sister
chromatids xx
Rad51…
Sequence
correction xx

2. Homology-directed gene repair
exogenous
repair substrate
I-SceI xx
sister
chromatids xx
Rad51K313Q
Sequence
correction xx

3. GFP reporter w/ exogenous donor
repair substrate
BcgI
CA
puroR
I-SceI
STOP
gene conversion
CA
GFP+
puroR
BcgI

4. DRGFP reporter puroR GFP+ puroR gene conversion CA CA I-SceI STOP
BcgI
gene conversion
CA
GFP+
puroR
BcgI
BcgI

5. 013108
Nalm6 Transfection
no DNA 24hr
1µg pmaxGFP 24hr

6. Nalm-6 DRGFP Functional screen of puroR cell lines
in 1?/24 I-SceI  nuc dependent GFP+

7. Nalm-6 DRGFP#13 72 hrs No I-SceI 72 hrs + I-SceI No TSA + TSA 030308

8. Possibilities Check more clones Chromatin Nalm-6 defect acetylation
methylation
Nalm-6 defect
Msh2-…
try other cell lines

9. David Bednarski
Amber Caracol
Jason Cummings
Luther Davis
Johanna Eddy
Nicolle Hamilton
Jana Mandic
Ellen Ordinario
Liz Sacho
Michael Stevenson
Aarthy Vallur
Munehisa Yabuki
Nancy Maizels

11. GFP x FL2-H
No DNA
+ pEGFP

12. New GFP reporter w/ exogenous donor
repair substrate
CMV
NeoR
lacO
intron
I-SceI…
STOP (x2)

13. Transfection Efficiency
48 hrs
72 hrs
96 hrs 10 1 2 3 4
FL2-H: FL2-Height
FL1-H: GFP 10 1 2 3 4
FL2-H: FL2-Height
FL1-H: GFP 10 1 2 3 4
FL2-H: FL2-Height
FL1-H: GFP No
pGFP 10 1 2 3 4
FL2-H: FL2-Height
FL1-H: GFP
29.3 10 1 2 3 4
FL2-H: FL2-Height
FL1-H: GFP
19.5 10 1 2 3 4
FL2-H: FL2-Height
FL1-H: GFP
13.3 +
pGFP

14. I-SceI Expression  Dead Cells?
No DNA
pEGFP
pCMV-Sce
200
400
600
800
1000
FSC-H: Forward Scatter
SSC-H: Side Scatter
83.1
0.81
200
400
600
800
1000
FSC-H: Forward Scatter
SSC-H: Side Scatter
83.2
1.23
200
400
600
800
1000
FSC-H: Forward Scatter
SSC-H: Side Scatter
76.3
3.19 48
hrs
030308

15. Nalm-6 DRGFP
GFP+
clone
No I-SceI
+ I-SceI
#13 72 hrs