Dicistronic Gene Expression in Developing Zebrafish

Fahrenkrug, Scott C.; Clark, Karl J.; Dahlquist, Mark O.; Hackett, Perry B.

doi:10.1007/pl00011810

Cited by 15 publications

(7 citation statements)

References 45 publications

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“…We have termed this -actin-apoaequorin-IRES-EGFP transgenic line of fish, -actinaeq. Use of the IRES-sequence enables the translation of both aeq and EGFP from a single mRNA, thus the expression level and distribution of EGFP reflects the expression level and distribution of apoaequorin (Jang et al, 1988;Fahrenkrug et al, 1999;Wang et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos

Cheung¹,

Webb²,

Love³

et al. 2011

Int. J. Dev. Biol.

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Intact zebrafish embryos were used as an in vivo animal model to investigate the role of Ca 2+ signaling during the differentiation of slow muscle cells (SMCs) within forming skeletal muscle. Transgenic zebrafish were generated using an     -actin promoter that targeted apoaequorin expression specifically to muscle cells. Supplementary Material (a movie and figure) for this paper is available at: http://dx

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Section: Resultsmentioning

confidence: 99%

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos

Cheung¹,

Webb²,

Love³

et al. 2011

Int. J. Dev. Biol.

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“…In addition, we wanted to monitor the gene expression at early stages of embryogenesis in order to quickly screen transgenic individuals. Hence, we placed an internal ribosome entry site (IRES; Fahrenkrug et al, 1999) and an open-reading frame (ORF) of green fluorescent protein (GFP) at 3′ downstream of the Actb-SLa (Fig. 1), so that the construct transcribes two ORFs into one mRNA from which SLa and GFP are simultaneously but separately translated (i.e., SLa is not tagged by GFP).…”

Section: Establishment Of the Sla-transgenic CI Medakamentioning

confidence: 99%

Effects of constitutive expression of somatolactin alpha on skin pigmentation in medaka

Fukamachi

Yada

Meyer

et al. 2009

Gene

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Somatolactin alpha (SLa) is a fish-specific peptide hormone secreted from the pituitary. Its functions have been investigated for the last decade but are still under debate. We previously reported a frame-shift mutation on SLa in a medaka mutant, color interfere (ci), which shows defects in body-color regulation, lipid storage, and cortisol secretion. In this study, we examined the effects of introducing a DNA fragment which constitutively expresses wild-type SLa under regulation of the beta-actin (Actb) promoter into the ci genome. We successfully visualized the transgene expression by taking advantage of an internal ribosome entry site (IRES) and the green fluorescent protein (GFP). The transgenic medaka, Actb-SLa:GFP, exhibited a reversed body-color phenotype of ci; more orange xanthophores and less white leucophores. We also detected more black melanophores and less silver iridophores, which indicates SLa's comprehensive role in regulating all types of pigment cells in the skin. Unexpectedly, the defects in lipid/cortisol contents remained in Actb-SLa:GFP. Therefore, the causal relationship between SLa and lipid/cortisol metabolisms relapses to an open question which needs to be reassessed by other types of experiments. Both the Actb-SLa:GFP and ci fish grow and mature similar to wild type, indicating SLa contributes little to growth regulation in spite of the fact that it binds to a teleost-specific paralog of growth-hormone receptor in vitro. The present study provides definitive evidence for SLa's principal and indispensable role in body-color regulation in medaka.

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“…IRES-based vectors are able to capture a wide range of genes expressed in a variety of tissues and embryos at different developmental stages [52]. The ECMV/IRES has been shown to function in developing zebrafish [63]. To determine the efficiency of the ECMV/IRES in driving the expression of reporter gene in our gene trap vector, we deleted the ECMV/IRES element from the pSPL3-Trap(intron) vector to generate the pSPL3-Trap(intron)ΔIRES (Figure 6A).…”

Section: Resultsmentioning

confidence: 99%

Effective Gene Trapping Mediated by Sleeping Beauty Transposon

Song

Long

et al. 2012

PLoS ONE

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Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 3′ bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous genes in vitro and in vivo.

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Dicistronic Gene Expression in Developing Zebrafish

Cited by 15 publications

References 45 publications

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos

Effects of constitutive expression of somatolactin alpha on skin pigmentation in medaka

Effective Gene Trapping Mediated by Sleeping Beauty Transposon

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