homology-directed gene repair
DESCRIPTION
Homology-directed gene repair. exogenous repair substrate. I-SceI. xx. sister chromatids. xx. Rad51…. Sequence correction. xx. Homology-directed gene repair. exogenous repair substrate. I-SceI. xx. sister chromatids. xx. Rad51 K313Q. Sequence correction. xx. BcgI. - PowerPoint PPT PresentationTRANSCRIPT
Homology-directed gene repair
Sequencecorrection
xxxx
xx
exogenousrepair substrate
Rad51…
sisterchromatids
I-SceI
Homology-directed gene repair
Sequencecorrection
xxxx
xx
exogenousrepair substrate
Rad51K313Q
sisterchromatids
I-SceI
GFP reporter w/ exogenous donor
puroR
I-SceI STOP
BcgI
puroR
BcgI
GFP+
gene conversion
exogenousrepair substrate
CA
CA
DRGFP reporter
puroR
I-SceI STOP BcgI
puroR
BcgI BcgI
GFP+
gene conversion
CA
CA
Nalm6 Transfection
0 200 400 600 800 1000Side Scatter
100
101
102
103
104
FL1-Height
0 2.27
88.79.02
0 200 400 600 800 1000Side Scatter
100
101
102
103
104
FL1-Height
0.89 30.7
60.28.17
no DNA 24hr 1µg pmaxGFP 24hr
013108
Nalm-6 DRGFP
• Functional screen of puroR cell lines
– in 1?/24 I-SceI nuc dependent GFP+
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.013
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
5.42e-3
Nalm-6 DRGFP#1372 hrs No I-SceI 72 hrs + I-SceI
No TSA
+ TSA
030308
Possibilities
• Check more clones
• Chromatin– acetylation– methylation
• Nalm-6 defect– Msh2-…– try other cell lines
David BednarskiAmber CaracolJason CummingsLuther DavisJohanna EddyNicolle HamiltonJana MandicEllen OrdinarioLiz SachoMichael StevensonAarthy VallurMunehisa YabukiNancy Maizels
GFP x FL2-H
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
29.3
0 200 400 600 800 1000FSC-H: Forward Scatter
100
101
102
103
104
FL1-H: GFP
00
1000
0 200 400 600 800 1000FSC-H: Forward Scatter
100
101
102
103
104
FL1-H: GFP
013.5
86.50
No DNA
+ pEGFP
New GFP reporter w/ exogenous donor
NeoR
I-SceI… STOP (x2)
exogenousrepair substrate
CMVintron
lacO
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1
-H:
GF
P
0
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1
-H:
GF
P
29.3
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1
-H:
GF
P
0
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1
-H:
GF
P
19.5
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1
-H:
GF
P
0
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1
-H:
GF
P
13.3
48 hrs 72 hrs 96 hrs
NopGFP
+pGFP
Transfection Efficiency
I-SceI Expression Dead Cells?
0 200 400 600 800 1000FSC-H: Forward Scatter
0
200
400
600
800
1000
SS
C-H
: S
ide
Sca
tter
83.2
1.23
0 200 400 600 800 1000FSC-H: Forward Scatter
0
200
400
600
800
1000
SS
C-H
: S
ide
Sca
tter
76.3
3.19
0 200 400 600 800 1000FSC-H: Forward Scatter
0
200
400
600
800
1000
SS
C-H
: S
ide
Sca
tter
83.1
0.81
No DNA pEGFP pCMV-Sce
48hrs
030308
Nalm-6 DRGFP
GFP+
clone No I-SceI + I-SceI