Abstract
Zebrafish transgenic lines provide valuable insights into gene functions, cell lineages and cell behaviors during development. Spatiotemporal control over transgene expression is a critical need in many experimental approaches, with applications in loss- and gain-of-function expression, ectopic expression and lineage tracing experiments. The Cre/loxP recombination system is a powerful tool to provide this control and the demand for validated Cre and loxP zebrafish transgenics is high. One of the major challenges to widespread application of Cre/loxP technology in zebrafish is comparatively small numbers of established tissue-specific Cre or CreERT2 lines. We used Tol2-mediated transgenesis to generate Tg(CrymCherry;-1.9mylz2:CreERT2) which provides an inducible CreERT2 source driven by muscle-specific mylz2 promoter. The transgenic specifically labels the trunk and tail skeletal muscles. We assessed the temporal responsiveness of the transgenic by screening with a validated loxP reporter transgenic ubi:Switch. Further, we evaluated the recombination efficiency in the transgenic with varying concentrations of 4-OHT, for different induction time periods and at different stages of embryogenesis and observed that higher recombination efficiency is achieved when embryos are induced with 10 μM 4-OHT from 10-somites or 24 hpf till 48 or 72 hpf. The transgenic is an addition to currently available zebrafish transgenesis toolbox and a significant tool to advance muscle biology studies in zebrafish.
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Abbreviations
- ER:
-
Estrogen receptor
- TAM:
-
Tamoxifen
- 4-OHT:
-
4-Hydroxytamoxifen
- mylz2:
-
Fast skeletal muscle myosin light chain 2
- WISH:
-
Whole mount in situ hybridization
- hpf:
-
Hours post fertilization
- PTU:
-
Phenylthiourea
- F0:
-
Founder animals (injected)
- F1:
-
1st generation animals
- dpi:
-
Days post injection
- dpf:
-
Days post fertilization
- EGFP:
-
Enhanced green fluorescent protein
- CTF:
-
Corrected total fluorescence
- SD:
-
Standard deviation
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Acknowledgements
We thank Dr. Jenna Galloway for discussions and providing valuable feedback towards the manuscript. We thank the Center for Computational and Integrative Biology (CCIB) at Massachusetts General Hospital for the use of the CCIB Core Facility (Cambridge, MA), which provided Sanger DNA sequencing service. This work was supported by the National Institute of General Medical Sciences (NIGMS) P01GM061354, and a research fellowship to K.M. from Shriners Hospitals for Children (Grant No. 84309).
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Mukherjee, K., Liao, E.C. Generation and characterization of a zebrafish muscle specific inducible Cre line. Transgenic Res 27, 559–569 (2018). https://doi.org/10.1007/s11248-018-0098-6
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DOI: https://doi.org/10.1007/s11248-018-0098-6