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Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.

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.

SUBMITTER: Bobis-Wozowicz S

PROVIDER: S-EPMC3983605 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.

Bobis-Wozowicz Sylwia S Galla Melanie M Alzubi Jamal J Kuehle Johannes J Baum Christopher C Schambach Axel A Cathomen Toni T

Scientific reports 20140411

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 ...[more]

PMID: 24722320

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Project description:Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of human ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in tumour cell lines. The hypothesis tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two tumour cell lines (H460 and HCT116). All four lines had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p=0.002) and 4.3±0.8% (p=0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no effect on glycolysis as measured by glucose consumption or lactate formation under oxic or anoxic conditions, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis. Use Affymetrix microarrays to examine the effecs of knockout of the gene ADPGK using Zinc Finger nucleases in two cultured cell lines: (i) HCT116 cells (three wild type cultures compared to three ADPGK-knockout cultures), (ii) H460 cells (two wild type cultures compared to two ADPGK-knockout cultures). Ten microarrays in total.

Dataset Information

Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.

Publications

Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.

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