Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters

Abstract

Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 α promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.

A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri)

“…EGFP expression continued throughout adulthood as determined at the age of 7 months (not shown). This is in agreement with recent studies in zebrafish and medaka showing EGFP expression under the control of different b-actin promoters to be active in numerous tissues of adult fish (Burket et al, 2008;Cho et al, 2011). In this particular experiment we obtained four potential founders from 350 injected oocytes indicating a very low efficiency of around 1%.…”

“…The EGFP expression was strong and could be observed as early as at the one-cell stage ($2 hpf), suggesting maternal deposition of EGFP protein and/or mRNA ( Fig. 4A; Burket et al, 2008). In all analyzed F1 embryos, EGFP expression continued to be strong during embryonic development (Fig.…”

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

“…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.…”

“…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.…”

Reverse genetics tools in zebrafish: A forward dive into endocrinology

“…It is a natural course of progress to apply the Cre/loxP system to the zebrafish model system to achieve the spatiotemporal control of gene expression, but shortage of a promoter/enhancer that ubiquitously drive expression of transgenes has been an obstacle [21,49]. Although several elements that are assumed to have broad control of genes have been identified, including h2afx, tbp and β-actin promoter, these expression driving elements are inactivated during the development and thus cannot be a proper ubiquitous promoter [3].…”