Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Abstract

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFNmediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.live imaging | quantitative biology

An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)

“…Previous studies have reported that simultaneous cleavage of the bait sequences of a donor plasmid and a genomic target site by targetable nucleases can improve the targeted integration efficiency of the plasmid [13, 16]. In our experiments, donor plasmids cleaved by bait systems also showed higher integration efficiencies than that of a donor plasmid with no targeted cleavage (Fig.…”

Stem Cell Imaging: From Bench to Bedside

“…Although the use of reporter gene imaging for long-term cell tracking in humans is currently restricted by regulatory hurdles, reservations about safety may be addressed by the use of human endogenous versions of reporter genes (e.g., human ferritin reporter for MRI (Campan et al, 2011) and human mitochondrial thymidine-kinase-2 for PET (Ponomarev et al, 2007). To avoid the problem of insertional mutagenesis, safety can be further enhanced by applying newer methods for genomic manipulation such as site-specific integration using phage integrases (Karow et al, 2011; Lan et al, 2012) and safe harbor mediated integration by zinc-finger nuclease (ZFN) (Ochiai et al, 2012; Wang et al, 2012), transcription activator-like effector nuclease (TALEN) (Sommer et al, 2014), or clustered regularly interspaced short palindromic repeats (CRISPR) (Yang et al, 2013). …”

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

“…This could be achieved by flanking donor sequences on the injected plasmid by nuclease target sites. Such an approach has been tested in sea urchin and was found to favor HDR in sea urchin embryos injected with ZFNs (Ochiai et al 2012). The use of a circular donor DNA with a nuclease site for in situ linearization but without any homology arms has also been shown recently to result in high-efficiency homology-independent knock-in of in vitro-transfected cells (Cristea et al 2013;Maresca et al 2013) and very recently in zebrafish (albeit concatemers were also observed using this strategy) (Auer et al 2014).…”