Abstract:
:Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Bobis-Wozowicz S,Galla M,Alzubi J,Kuehle J,Baum C,Schambach A,Cathomen Tdoi
10.1038/srep04656subject
Has Abstractpub_date
2014-04-11 00:00:00pages
4656issn
2045-2322pii
srep04656journal_volume
4pub_type
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