Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

Abstract

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

“…The advent of genomic-targeted modification technology mediated by ZFN, TALENs, and CRISPR/Cas9 gives hope to solving this technical problem. Cells or embryos of the mouse, zebrafish, other model organisms, and certain economic species have had ZFN and TALENs successfully applied to achieve site-directed mutagenesis of endogenous genes (Doyon et al 2008;Kim et al 1996;Osiak et al 2011). The CRISPR/Cas9 system has a wide range of potential applications in gene editing at the cellular level and the construction and screening of large-scale mutant libraries due to the characteristic short palindromic repeat sequences (Cermak et al 2011;Li et al 2011).…”

“…Targeted genome modification technology can be mediated by zinc finger nucleases (ZFNs) (Doyon et al 2008;Kim et al 1996;Osiak et al 2011), transcription activator-like effector nucleases (TALENs) (Bogdanove and Voytas 2011;Huang et al 2011;Sander et al 2011), and type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) (Cong et al 2013;Hwang et al 2013). TALENs are artificial nucleases that consist of DNA binding domains of transcription activator-like (TAL) effector and catalytic Fok I domains (Bogdanove and Voytas 2011).…”

Efficient Detection, Quantification and Enrichment of Subtle Allelic Alterations

“…12 The ability to efficiently identify subtle allelic alterations is essential in these ZFN- and TALEN-mediated GT experiments. In this study, we have developed TAGE and PAGE in model systems and a real GT experiment as versatile procedures for detecting allelic variations in ordinary laboratories without specialized equipment and devices.…”

Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants

“…12.2a). ZFNs have been used to create targeted DSBs and enable genome modification in a broad spectrum of genomes, including human (Lombardo et al 2007;Moehle et al 2007;Perez et al 2008;Porteus and Baltimore 2003;Provasi et al 2012;Sebastiano et al 2011;Urnov et al 2005;Wilen et al 2011), hamster (Santiago et al 2008), mouse (Osiak et al 2011), pig (Hauschild et al 2011, frog (Young et al 2011), zebra fish (Doyon et al 2008), insect (Beumer et al 2006;Bibikova et al 2002), roundworm (Morton et al 2006), and Plasmodium (Straimer et al 2012). The present chapter reviews the use of designed ZFNs for inducing targeted DSBs and facilitating genome modification in plants ( …”