homology-directed gene repair sequence correction xx exogenous repair substrate rad51… sister...
TRANSCRIPT
Homology-directed gene repair
Sequencecorrection
xxxx
xx
exogenousrepair substrate
Rad51…
sisterchromatids
I-SceI
Homology-directed gene repair
Sequencecorrection
xxxx
xx
exogenousrepair substrate
Rad51…
sisterchromatids
I-SceI
DRGFP reporter
puroR
I-SceI STOP BcgI
puroR
BcgI BcgI
GFP+
gene conversion
CA
CA
293T stable DRGFP (S8)
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.058
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.063
no DNA pCMV-I-SceI
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.033
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.084
48 hrs
72 hrs
041408
Direct Repeat Reporters:recombination in cis
SceISTOPpDRGFP
andpDRGFP-lacO
pCMV-I-SceI
293T DRGFP (cis)
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
1.76e-3
I-SceI
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
1.99
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
10.9
DRGFP+I-SceIDRGFP
041408
293T DRGFP-lacO (cis)
041408
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.32
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
6.05
DRGFPlacO DRGFPlacO+I-SceI
Exogenous Donor Reporter:recombination in trans
SceISTOPpZFGFP
andpZFGFP-lacO
pKS-GFP
pCMV-I-SceI
293T ZFGFP + iGFP (trans)
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.15
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
5.16
ZFGFP+iGFP ZFGFP+iGFP+I-SceI
041408
293T ZFGFP-lacO + iGFP (trans)
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
0.13
100 101 102 103 104
FL2-H: FL2-Height
100
101
102
103
104
FL1-H: GFP
1.18
ZFGFP-lacO ZFGFP-lacO+iGFP+I-SceI
041408
Too Much DNA?
293T: Efficiency of Transient Transfection
050208
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
0
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
88.2
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
97.2
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
99.9
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
99.7
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
83.2
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
0.038
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
0.16
500 ng eGFP
no DNA
1.0 g eGFP 2.0 g eGFP 4.0 g eGFP
62 ng eGFP 125 ng eGFP 250 ng eGFP
“Stable” DRGFP?
“Stable” DRGFP (puroS)
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
4.5e-3
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
3.35e-3
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
0.014
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
0.05
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
0.024
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
0.014
No DNA
CMV-Sce
48 hrs 73 hrs 98 hrs
“Stable” DRGFP (puroR)
No DNA
CMV-Sce
48 hrs 73 hrs 98 hrs
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
2.29
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
2.05
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
2
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
2.93
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
3.34
100 101 102 103 104
FL2-H
100
101
102
103
104
GFP
3.07
Plans
• Lines with integrated trans reporter– Enhance repair by tethering chromatin
modifiers and repair factors
• Back to B-cells