Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish

Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein (EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adenoassociated virus (AAV) and driven by zebrafish cw-actin (p alpha -actin-EGFP-ITR) or medaka p-actin promoters (p beta -actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the p alpha -actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the p beta -actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from p alpha -actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from p beta -actin-EGFP-ITR-injected embryos crossed with wildtype fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both or-actin and p-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish. (C) 2001 Wiley-Liss, Inc.