Conditional expression of a myocardium‐specific transgene in zebrafish transgenic lines

Abstract

To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTAresponsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage. Developmental Dynamics 233:1294 -1303, 2005.

Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish

“…They allow for tissuespecific, inducible, reversible and tunable control of gene expression in embryos and adults. Although other systems for conditional gene expression have been adapted for use in zebrafish, such as the Cre-Lox system or previous installments of the TetON system (6)(7)(8)10), the main advantages of the systems presented here are their reversibility and very low leakiness. Thus, they will be particularly useful for applications where reversibility is desired, for example for manipulation of pathways that have temporally distinct roles in the same tissue, or for studying the effects of transient manipulation of progenitor cell pools, for example during regeneration.…”

“…In the mouse, conditional and reversible tissue-specific gene expression can be achieved using the TetON system, which combines tissue-specific transgenic expression of a tetracycline (Tet)-or doxycycline (Dox)-inducible transcriptional activator (TetActivator) with a Tet-responsive transgene (9). The applicability of this system to zebrafish has been reported, but its usefulness was confounded by leakiness and lack of reversibility (10). Here, we present two modified, dually inducible TetON systems, which significantly reduce leakiness and display improved activity, and show that such systems can be used to achieve conditional, tissue-specific, and reversible gene expression in embryonic and adult zebrafish.…”

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

“…The Tet-controlled systems are the most popular and reliable methods which are used to modulate the expression of genes in cultured cells and organisms such as Drosophila and mice (Stebbins et al, 2001). A transgenic line has been developed for heart-specific expression of rtTA gene under the control of a clmc2 promoter (Huang et al, 2005). This transgenic line is very useful for the study of gene function during heart development and disorders, but lacks clear background in terms of rtTA gene integration.…”

“…The integration of multiple transgene copies at a distinct locus or in different chromosomes, is always associated with the disruption of endogenous gene expression or regulation, and thus affects embryonic development and normal physiological functions (Livet et al, 2007). However, most transgenic fish lines currently available are not well-characterized in terms of integration site, stable expression and transmission of foreign genes (Huang et al, 2005). In this study, we had obtained an rtTA-transgenic zebrafish line that carried a single copy of the rtTA gene in the genome and exhibited no defects in embryonic development and hatching rate.…”

“…The Tet-on system was first used in zebrafish for improving GFP expression in a gene-trap vector. A transgenic zebrafish line with heart-specific expression of rtTA-M2 was later generated and a dose-dependent GFP expression by Dox-induction was detected in the hearts of transgenic larvae (Huang et al, 2005). Thus, the Tet-on system seems to be an invaluable tool for investigation of gene function and regulation under both physiological and pathological conditions.…”