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

Parts used

Key findings

• A 3.5 kb fragment immediately upstream of the ATG start codon in exon 2 of the zebrafish ubiquitin B gene (zgc:172187, linkage group 5), including the putative intron/exon splice junctions, contains sufficient regulatory sequence to drive strong transgene expression. The locus has a 71 bp non-coding first exon, a 2 kb intron and a second exon encoding an eight-repeat ubiquitin peptide precursor, a structure homologous to ubiquitin loci in other species.
• The zebrafish ubi promoter begins driving transgene expression at the mid-blastula transition, roughly 2.75 hours post fertilization. Tol2-mediated injection of a −3.5ubi:EGFP reporter into one-cell-stage embryos gives detectable mosaic EGFP within 4 hours, consistent with active histone marks appearing at the ubi locus at that transition, and germline-transmitted insertions show EGFP in all blastula cells by 4 to 5 hours post fertilization.
• Transgenes driven by the zebrafish ubi promoter are maternally deposited and show allele dosage effects. Outcrossing heterozygous ubi:EGFP females to wild-type males gives embryos with strong maternal EGFP contribution detectable at the eight-cell stage, and incrossing heterozygous parents produces roughly 25% of embryos with notably more intense fluorescence, subsequently confirmed as homozygous for the transgene.
• The zebrafish ubi promoter drives expression in tissues that other putatively ubiquitous zebrafish promoters fail to reach. Heterozygous ubi:EGFP adults show homogeneous EGFP in all externally visible structures including the fins — tissues not labelled by ef1α, tbp or β-actin driven reporters — and anti-EGFP immunohistochemistry on sagittal sections reveals expression across many internal organs, including retina and erythrocytes, which had remained untargeted by other ubiquitous promoters.
• ubi promoter activity is near-ubiquitous but not strictly uniform: immunohistochemistry identifies small regions in internal organs where EGFP levels are substantially lower than in adjacent tissue, including the cell bodies of subsets of central nervous system neurons and the columnar epithelial cells of kidney tubules.
• The zebrafish ubi promoter expresses in every resolvable blood cell lineage, unlike the β-actin promoter conventionally used to mark donor cells in zebrafish transplantation. Flow cytometry of whole kidney marrow from adult ubi:EGFP and ubi:mCherry heterozygotes shows robust fluorophore expression in the vast majority of cells across erythroid, myeloid and lymphoid gates plus precursor populations, whereas β-actin:EGFP transgenics express weakly in hematopoietic lineages and barely at all in erythrocytes.
• Because ubi-driven fluorophores mark all blood lineages, they permit true multilineage engraftment to be scored after hematopoietic transplantation. Retro-orbital transplantation of whole kidney marrow from ubi:mCherry or ubi:EGFP adults into irradiated transparent casper (roy−/−; nacre−/−) recipients gives strong localized fluorescence over the kidney territory by three weeks, with flow cytometry showing chimeric donor contribution to every light-scatter-resolvable blood population. Fluorophore intensity is retained in long-term transplants beyond three months and in competitive transplants mixing ubi:EGFP and ubi:mCherry donor populations.
• A Tg(−3.5ubi:creERt2; cmlc2-EGFP) transgenic line provides ubiquitous Cre activity that is strictly dependent on 4-hydroxytamoxifen. Crossed to lmo2:loxP-dsRed-loxP-EGFP reporter females and induced with 5 μM 4-OHT from shield stage, double-positive embryos show strong mosaic EGFP in vasculature and hematopoietic system by 24 hours post fertilization, while ethanol-treated controls never show detectable GFP. The same 4-OHT dependence holds with the eab2:[EGFP-T-mCherry] (FlEX) inversion reporter, where 4-OHT at shield stage triggers strong mosaic mCherry by 3 days post fertilization and solvent controls produce none.
• Cells recombined by ubi-driven CreERt2 in the lmo2:Switch reporter are always single-positive for either EGFP or mCherry rather than both, indicating that 4-OHT activity — and therefore the probability of a loxP recombination event — fades across the window between induction at shield stage and the onset of lmo2 reporter expression.
• Tg(−3.5ubi:loxP-GFP-loxP-mCherry), or ubi:Switch, is a lineage-tracing reporter whose expression persists at all stages because the ubi promoter drives it. Crossed to ubi:creERt2 and induced with 5 μM 4-OHT at shield stage, double-positive embryos show strong widespread mCherry by 3 days post fertilization that intensifies over time as EGFP fades, while ethanol-treated and creERt2-negative siblings remain EGFP-only.
• 4-hydroxytamoxifen enters zebrafish embryos rapidly enough for pulse-based temporal control of lineage tracing: a 15-minute exposure to 5 μM 4-OHT followed by thorough washing still produces strong, widespread mCherry mosaicism in ubi:creERt2; ubi:Switch embryos, though less complete than continuous exposure. This supports using temporal 4-OHT pulses to trace distinct cell populations, which is technically difficult in mouse development.
• loxP recombination by ubi-driven CreERt2 is 4-OHT concentration dependent, with a sharp threshold. Reading out at 30 hours post fertilization after induction at bud stage, 5 μM 4-OHT gives the strongest recombination, 1 μM gives a notably weaker and more mosaic response, and 0.5 μM fails to trigger widespread mCherry expression at all.
• ubi-driven CreERt2 remains active across a broad developmental window, with induction later in development giving progressively more mosaic recombination. Embryos induced at 24 hours post fertilization show slightly lower mCherry mosaicism and weaker levels at 3 days post fertilization than shield-stage siblings, intensifying over time as EGFP fades, and a cohort induced at 6 days post fertilization shows widespread mCherry by 9 days with only faint expression in the head. Three factors explain this: more cells must be independently recombined at later stages, mCherry has less time to accumulate, and 4-OHT tissue penetration is limited in growing larvae, particularly in the head.
• Different loxP switch transgenes respond with different sensitivity to the same CreERt2 source, likely reflecting genomic integration site and loxP accessibility rather than Cre activity — which is why building a new switch line on the ubi promoter was necessary to obtain efficient, position-effect-free recombination.
• Promoter cross-talk can occur between a transgene's selection marker and its primary cassette: ubi:creERt2; ubi:Switch larvae induced at 24 hours post fertilization show strong mCherry in the heart, the territory where the linked cmlc2-EGFP marker driver is active.

Abstract

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:cre(ERt2) transgene, as well as ubi:loxP-EGFP-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.