title: Novel reporter systems for facile evaluation of I-SceI-mediated genome editing.
abstract: Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects.
authors: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors: Nina M
authors: NM
authors: Muñoz
authors: Brian C
authors: BC
authors: Beard
authors: Byoung Y
authors: BY
authors: Ryu
authors: Ralf M
authors: RM
authors: Luche
authors: Grant D
authors: GD
authors: Trobridge
authors: David J
authors: DJ
authors: Rawlings
authors: Andrew M
authors: AM
authors: Scharenberg
authors: Hans-Peter
authors: HP
authors: Kiem
mesh_terms: D002460:Cell Line; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D016678:Genome; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D011496:Protein Methyltransferases / Q000032:analysis / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011496:Protein Methyltransferases; D004706:Endodeoxyribonucleases