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