Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena

Abstract

Marine medaka Oryzias dancena, a candidate model organism, represents many attractive merits as a material for experimental transgenesis and/or heterologous expression assay particularly in the field of ecotoxicology and developmental biology. In this study, cytoskeletal β-actin gene was characterized from O. dancena and the functional capability of its promoter to drive constitutive expression of foreign reporter protein was evaluated. The O. dancena β-actin gene possessed a conserved genomic organization of vertebrate major cytoplasmic actin genes and the bioinformatic analysis of its 5'-upstream regulatory region predicted various transcription factor binding motifs. Heterologous expression assay using a red fluorescent protein (RFP) reporter construct driven by the O. dancena β-actin regulator resulted in stunningly bright expression of red fluorescence signals in not only microinjected embryos but also grown-up transgenic adults. Although founder transgenics exhibited mosaic patterns of RFP expression, transgenic offspring in subsequent generations displayed a vivid and uniform expression of RFP continually from embryos to adults. Based on the blot hybridization assays, two transgenic lines established in this study were proven to possess high copy numbers of transgene integrants (approximately 240 and 34 copies, respectively), and the transgenic genotype in both lines could successfully be passed stably up to three generations, although the rate of transgene transmission in one of the two transgenic lines was significantly lower than expected Mendelian ratio. Significant red fluorescence color could be ubiquitously observable in all the tissues or organs of the transgenics. Quantitative real-time RT-PCR represented that the expression pattern of transgene under the regulation of β-actin promoter would resemble, in overall, the regulation of endogenous β-actin gene in adult tissues, although putative mechanism for competitive or independent regulation between transgene and endogenous gene could also be found in several tissues. Results from this study undoubtedly indicate that the O. dancena β-actin promoter would be powerful enough to fluorescently visualize most cell types in vivo throughout its whole lifespan. This study could be a useful start point for a variety of transgenic experiments with this species concerning the constitutive expression of living fluorescent color reporters and other foreign proteins.

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%.…”

“…Analysis of EGFP expression revealed that all four larvae showed a mosaic expression pattern of EGFP as it has been frequently described for the founder generation of transgenic animals ( Fig. 3C,D ;Winkler et al, 1991;Hosemann et al, 2004;Cho et al, 2011). EGFP expression continued throughout adulthood as determined at the age of 7 months (not shown).…”

Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

“…The functional ability of the β-actin regulator to drive ubiquitous expression of the red fluorescent reporter in this fish species has been described previously (Cho et al, 2011). Details of the expression patterns of the endogenous β-actin gene, as well as the β-actin promoterdriven RFP expression in marine medaka, were provided in our previous work (Cho et al, 2011). The β-actin-driven fluorescent signal in transgenic marine medaka usually becomes clearly visible by the embryonic stage (onset often occurs at late gastrulation), with the RFP phenotype lasting persistently to adulthood.…”

“…Various transgenic marine medaka founders were generated by the microinjection of a fluorescent reporter construct consisting of the RFP gene (DsRed2; Clontech Laboratories Inc., Mountain View, CA, USA) under the control of endogenous β-actin regulator (podβ-actRFP; Cho et al, 2011). Microinjection was performed on one-cell embryos using the ClaI-linearized podβ-actRFP construct suspended at a concentration of 50 μg/ml.…”

“…Microinjection was performed on one-cell embryos using the ClaI-linearized podβ-actRFP construct suspended at a concentration of 50 μg/ml. The functional ability of the β-actin regulator to drive ubiquitous expression of the red fluorescent reporter in this fish species has been described previously (Cho et al, 2011). Details of the expression patterns of the endogenous β-actin gene, as well as the β-actin promoterdriven RFP expression in marine medaka, were provided in our previous work (Cho et al, 2011).…”