271 Expression of IGF1 Receptor Ribozyme and IGFBP3 To Inhibit Rat Glioma Cell Growth 270 Gene Transfer of an Engineered Transcription Factor Promoting Expression of VEGF A Accelerates Rehabilitation[.]
269 Flow Cytometric Analysis of DNA Binding and Cleavage by Cell Surface-Displayed Homing Endonucleases Petra Volna.l-' Jordan Jarjour,2.3 Sarah Baxter,' Barry L Stoddard,' Raymond J Monnat, Jr,' Andrew M Scharenberg.P? 'Pediatrics University of Washington , Seattle WA; 2Immllllolog); Children s Hospital and Regional Medical Center; Seattle, IVA; 'Immunology; University ofWashington, Seattle WA; 'Division 0/ Basic Sciences Fred Hutchinson Cancer Research Center; Seattle /I'iI ; :;Pathology University of Washington, Seattle /I'iI LAGLtDADG homing endonucleases (LHE)s cleave 18-24 base pair DNA sequences and are promising enzymes for applications requiring sequence specific DNA cleavage amongst genome-sized backgrounds We developed a method for cell surface display of LHEs which facilitates analysis oftheir DNA binding and cleavage properties using flow cytometry.Cells expressing surface Lt-I Es can be stained with fluorescently conjugateddouble-strandedoligonucleotides (dsOligos) containing their respective target sequences The signal is absolutelysequencespecificand undetectablewith dsOligos carrying single base pair substitutions [Figure I] LHE-dsOligo interactions facilitate rapid enrichment and viable recovery ofrare LHE expressing cells by both FACSand MACS.Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNAcleavage As recapitulation of DNA binding and cleavage by surface displayed Ll-lEsprovides a high-throughput approach to library screening, we have explored using LI-IE surface expression in combination with somatic hypermutation as a means for iterative selection of variants with novel sequence recognition specificities Data from this approach and future directions will be discussed Staining ofeell Surface HEs willi dsOligos: I·AnII express ing clones H-OreI express ing done 270 Gene Transfer of an Engineered Transcription Factor Promoting Expression of VEGF-A Accelerates Rehabilitation of the Crushed Recurrent Laryngeal Nerve S Brett Heavener,' Kevin Fung ! S Kaye Spratt,' Casey C Case,' Laurel E Roberts,' Norman D Hogikyan,' Eva L Feldman.' 'Medical Center; University ofMichigan, Ann Arbor, M/; 20tolar- yngology; University 0/ Western Ontario, London, ON, Canada; 'Development Sangamo Biosciences, Richmond CA Vocalfold motion impairmentmay result from recurrent laryngeal nerve (RLN) injury or degeneration and is a frequent yet unsolved problem faced by the otolaryngologist, In addition to dysphonia, RLN injury may lead to aspiration , and airway compromise is Molecular Therapy Volume '5 Supplement I ,\ by 2