High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin
No transgenic zebrafish had used a zebrafish tissue-specific promoter for reliable reporters. This paper isolated zebrafish actin promoters to generate GFP transgenic lines. 40 of 41 alpha-actin-GFP lines expressed GFP specifically in muscle, at up to 21% transgenic frequency.
Parts used
Key findings
- The zebrafish muscle-specific α-actin promoter (3.9 kb) was used to drive GFP expression in transgenic zebrafish. A total of 41 GFP-expressing lines were generated at 21% frequency (41 of 194 injected), with 40 of 41 lines showing uniform, muscle-specific GFP expression — the first demonstration that a zebrafish tissue-specific promoter reliably controls transgene expression in stable germline-transmitted lines.
- The zebrafish cytoskeletal β-actin promoter (~17 kb) drove GFP expression throughout the whole body in all 4 transgenic lines generated. Pan-body expression mirrored endogenous β-actin distribution, demonstrating that zebrafish-origin ubiquitous promoters enable reproducible, whole-body reporter gene expression without the variegation seen with heterologous viral promoters in prior studies.
- Upstream plasmid vector sequence adjacent to the transgene reduced GFP expression levels in stable lines. Transient GFP expression in injected embryos did not predict germline transgenic frequency, and co-injection of SV40 NLS peptide showed no significant improvement in transgenic efficiency — identifying three technical variables that influence expression but not germline transmission rates.