Lentiviral Design for Delivery of Homing Endonucleases mCherry HA-NLS-HE RSVR U5 hPGK R U5sinU3 Repair Template Cppt RRE RRL Lentiviral Backbone.

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Presentation transcript:

Lentiviral Design for Delivery of Homing Endonucleases mCherry HA-NLS-HE RSVR U5 hPGK R U5sinU3 Repair Template Cppt RRE RRL Lentiviral Backbone

Integrase Deficient Lenti Virus psPAX2 D64V untreated1:101:501:1001:5001:1000 DAY 2DAY 12DAY 8 DAY 5 Day 19 post transduction: 0.62% of cells remain GFP+

Directions Effect of local chromatin structure on the efficiency of cutting and repair Role of repair template topology on conversion frequency

HEs for Targeted Integration Can an HE induced double stranded break facilitate targeted transgene integration? Genomic -7C+11C Sce Site in Canine

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: recSce cleaves +11C miscognante linearized 5units0.5units 0.2units

Directions Characterize -7C-Sce cleavage of the target site using DR-reporter Assess cleavage of the genomic site in-vivo Attempt to target reporter to the locus

2 nucleotide changes switch fluorophores Exogenous Template HDR Blue-Green Reporter System Swappable HE cleavage site 70bp from target, does not affect reporter Intron allows use of >1Kb repair template T Y G Fluorophore “Switch” Repair Template Asc1Age1 eGFP T Y G eGFP Repair Product S H G Asc1Age 1 eBFP 1-52 eBFP Intron 70 bp eBFP HE Substrate Swappable HE substrate