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WT-7C+11C -7C +11C Ani WT-7C+11C -7C +11C Ani WT-7C+11C -7C +11C Ani WT-7C+11C -7C +11C Ani Substrate: In-Vitro recSce Cleavage of Canine Site linearized 5units0.5units 0.2units An endogenous intergenic 2-off Sce site (-7C +11C) in the Canine genome is a potential safe harbor site for transgenes. Linearized plasmid DNA containing the indicated targets was digested with recombinant I-SceI to assess cleavability.
Citation preview
NGEC Applications Meeting05-06-08
Mike CertoScharenberg Lab
Transient DR-GFP Assay
WT -7C +11C -7C+11C
Ani WT -7C +11C -7C+11C
Ani WT -7C +11C -7C+11C
Ani WT -7C +11C -7C+11C
AniSubstrate:
In-Vitro recSce Cleavage of Canine Site
linearized 5units 0.5units 0.2units
An endogenous intergenic 2-off Sce site (-7C +11C) in the Canine genome is a potential safe harbor site for transgenes. Linearized plasmid DNA containing the indicated targets was digested with recombinant I-SceI to assess cleavability.
Transient DR-GFP Assay for In-vivo Enzyme/Target Discrimination
GFP control Sce DR alone Sce DR + I-SceI
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
Untreated
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-SceWT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
WT-DR
-7C-DR
+11C-DR
-7C+11C-DR
WT-Sce -7C-Sce
I-AniI vs Y2 In-Vivo
untreated Ani-DR Ani-DR + Ani Ani-DR + Ani-Y2
I-SceI is Intolerant of C-terminal Fluorescent Protein Fusions
- I-SceII-SceI
-mCherryI-SceI-3xG4S
-mCherryI-SceI-IRES
-mCherry
Sce DR-GFP
Transient DR assay to assess functionality of Sce-Cterm fusions
Conclusions: Transient DR-GFP
Assay
• Can be used to quickly discern activity between enzyme variants and targets.
• Signal can be adjusted by varying DNA input.
• Provides information in the context of mammalian gene conversion.
Experimental
• -7C + 11C Canine target will likely require enzyme optimization
• WT I-AniI induces gene conversion at suboptimal rates
• Y2 variant performs similar to I-SceI • I-SceI is non-permissive of C terminal fluorescent fusions, but IRES allows for enzyme function and detection
Blue-Green Color Change
• 2 nucleotide changes switch fluorophores
Trans-HDR Blue-Green Reporter
• Swappable HE cleavage site• Intron allows use of >1Kb repair template
T Y Fluorophore “Switch” Repair Template
Asc1 Age1eGFP53-238
T Y eGFP Repair Product
S H
Asc1
Age1eBFP1-52 eBFP53-238
Intron
70 bp
eBFP HE Substrate
Swappable HE substrate
Lenti Viral Vector for Color Change Reporter
pRRL SFFV eBFP IRES Puro Sce WPRE9215 bp
AmpRGag (SL4)
PuroR
eBFP N-term
eBFP
mouse IL2Rgamma Intron 4
RU5
U3*RU5
SFFV
PBS (SL123)
RREcPPT
WPRE
PolyA
IRES
SD
YTRAY Branch point
SAI-Sce target site
RSV
F1 ORISV40 pA/ORI
Nef*
S65 H66
Color Change Reporter Transduction
100,000 293T cells transduced with either RRL eGFPsce reporter or RRL eBFPsce reporter. Based on eGFP positive cells at 1uL of viral supernatant, titer is estimated at ~ 2 x10 I.U./ml giving M.O.I ~ 0.5 . This should yeild a population averaging 1 integration per cell.
7
Single Cell Sorting
We gated on increasingly stringent populations (3% to 0.5%) and single cell sorted into 96 wells plates. As clones come up, we will re-analyze by flow for expression, and conduct southern blots to determine single cell integrants.
Integrated Reporter Is Unspliced
acro
ss in
tron
within
intro
n
Across IntronNon-spliced product yeilds: 1300bpSpliced product yeilds: 720bp
Within IntronNon-spliced product yeilds: 1140bpSpliced product yeilds: no band
1500bp
1000bp
No unspliced products were detected
PCR of Genomic DNA Following Transduction
untre
ated
Sce
I-SceI Digestion of gDNA PCR Product
1500bp
1000bp
I-SceI site is cleavable
The PCR product of the integrated reporter was digested with recombinant Sce to ensure the presence of a functional target
untreated repair template alone sce expression alone Sce + repair template
Gene Conversion
Polyclonal population of puro selected cells were transfected with a plasmid containing I-Sce expression and an eGFP repair template. Cells were FACS 5 days post transfection.
Effect of Homology and Track Length on Conversion in Trans
untreated repair SceSce + repair 1uG
Sce + repair 2uG
Sce + repair 5uG
untreated repair SceSce + repair 1uG
Sce + repair 2uG
Sce + repair 5uG
eBFP
azB
FP
Disparity of Gene Conversion at Distinct Loci
Single cell clones from the polyclonal population were subjected to gene conversion assay
293T polyclonal
Single Cell Clones
IDLV Gene Conversion
~ 60,000nG p24
untreated repair alone Sce alone Sce + repair
Assay
• eBFP likely detectable as single integrant at particular loci
• Conversion rates of ~ 3%
• Blue to Green? Fluorescent protein stability an issue?
Conclusions: Blue-Green Color Change
Experiments
• Single cell clones exhibit differential conversion ability
• Low expression off IDLV contributes to inefficient repair