Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish

Christian Mosimann Charles K. Kaufman Pulin Li Emily K. Pugach Owen J. Tamplin Leonard I. Zon () SUMMARY Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:creERt2 transgene, as well as ubi:loxPEGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish. INTRODUCTION A ubiquitous promoter/enhancer to drive transgenes is a key component of a complete transgenesis toolkit in any model organism. Enhancer trap screening in the mouse previously identified the Rosa26 locus, which became the current gold standard for ubiquitous transgene expression and for Cre/loxPbased lineage tracing at all stages of development and in all adult tissues (Friedrich and Soriano, 1991; Soriano, 1999; Zambrowicz et al., 1997). The lack of a homologous tool in zebrafish has particularly limited the full potential of Cre/loxP-based lineagetracing approaches that complement the powerful imaging possibilities and genetic malleability of the model system. As genomic knock-in methods for zebrafish are currently missing, a broadly applicable ubiquitous transgene control element needs to be accessible in simple cloning vectors. Strong candidates for ubiquitous zebrafish promoters/enhancers are thus control regions of genes encoding factors required in fundamental cellular processes. Several native zebrafish as well as exogenous control elements have been previously deemed ubiquitous in vivo and successfully applied in genetic experiments; these include the zebrafish h2afx, tbp, versions of the -actin control elements (Burket et al., 2008; Gillette-Ferguson et al., 2003; Higashijima et al., 1997; Kwan et al., 2007), and the Xenopus laevis-derived elongation factor 1a promoter (XlEef1a1) (Johnson and Krieg, 1994; Kawakami et al., 2004). Each of these promoter fragments has shortcomings in different developmental stages and cell types. A common problem is their progressive inactivation during the course of development, particularly in parts of the nervous system, the fin fold, hematopoietic cell populations, or even ubiquitously in all tissues. For example, the commonly applied XlEef1a1 reportedly only expresses strongly during gastrulation and midsomitogenesis before being restricted to specific cell types, although prolonged expression has been reported for isolated transgenes (Collins et al., 2010; Hans et al., 2009). The -actin promoter fragments do retain expression in adults, yet show no significant activity in erythrocytes or fins, or several other cell types (Burket et al., 2008; Traver et al., 2003). This likely reflects specialization of differentiating cells, potential cell-type-specific requirements for basic molecular mechanisms controlling translation or chromatin maintenance, as well as transgene dependence on the genomic integration context. Lineage-tracing transgenes using currently available transgene promoters in zebrafish therefore do not allow complete cell-fate tracing to all descendant tissues nor lineage analysis over prolonged time periods. Furthermore, the extensive creation of loxP cassettebased transgenes requires a reproducible and reliable driver element with broad cloning compatibility. Seminal work by Hans et al. (Hans et al., 2009) established the functionality of tamoxifen (TAM)-inducible CreERt2-mediated loxP recombination (Feil et al., 1996; Feil et al., 1997) in zebrafish. The fast uptake and action of the active TAM metabolite 4-hydroxytamoxifen (4-OHT) triggers dose-dependent CreERt2-mediated loxP excision events within 2-4 hours of administration (Hans et al., 2009). As the drug is easily applied through simple addition to the embryo medium, 4-OHTinducible CreERt2 is a desirable tool for studying zebrafish development. However, to reliably and flexibly trace loxP excision events in cell-lineage experiments at all stages of development, or to trigger Gal4 and other genetic modifiers using tissue-specific CreERt2 sources, a ubiquitously expressed loxP switch construct driver is eagerly needed (Blackburn and Langenau, 2010; Hans et al., 2009). In Drosophila, the strongest and most faithful ubiquitous control element currently in use is derived from the 5 region of the ubiquitin (ubi) gene, which encodes the evolutionarily conserved multimeric Ubiquitin (Ubi) peptide precursor (Lee et al., 1988). ubi-driven green fluorescent protein (GFP) transgenes are used widely as reliable markers for various genetic applications at all stages of Drosophila development. The evolutionarily conserved ubi control region has also been cloned from maize and has found widespread application in plant transgenesis (Christensen et al., 1992). The human ubi promoter has been isolated and drives strong transgene expression in a variety of cell culture systems (Schorpp et al., 1996). Notably, UBI-GFP/BL6 transgenic mice express EGFP in virtually all cells of the hematopoietic system, thus greatly enhancing its utility in bone marrow stem cell transplantation experiments (Schaefer et al., 2001). Based on this precedent, we sought to identify and characterize a ubiquitin locus in zebrafish to generate an endogenously derived ubiquitous transgene driver. Analogous to the reported Drosophila, maize, and human loci, we cloned a 3.5 kb 5 region upstream of the zebrafish ubiquitin B translational start site spanning the proximal upstream region, the first noncoding exon, and the only intron of the gene. In transient injections into zebrafish embryos, the ubi control region drives strong and ubiquitous visible expression of an EGFP reporter gene within 4 hours post-injection, likely starting at the onset of zygotic transcription. Stable ubi:EGFP lines reveal strong expression in all analyzed external and internal organs, including the retina, fin fold, and across all blood cell types from embryo to adulthood. The blood expression of ubi-driven fluorophores enabled us to perform adult zebrafish transplants with whole kidney marrow (WKM), which harbors the adult hematopoietic stem cells, into irradiated recipients and to monitor true multilineage repopulation from the transplanted cell populations. We further created ubi:creERt2 tra (...truncated)