Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system

• Published in: Transgenic research Vol. 14; no. 2; pp. 217 - 223
• Main Authors: XIUFANG PAN, HAIYAN WAN, CHIA, Wendy, YAN TONG, ZHIYUAN GONG
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer 01.04.2005; Springer Nature B.V
• Subjects: Animals; Animals, Genetically Modified; Base Sequence; Biological and medical sciences; Biotechnology; Danio rerio; DNA - analysis; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genetic engineering; Genetic Markers; Genetic technics; Green Fluorescent Proteins - genetics; Integrases - genetics; Methods. Procedures. Technologies; Molecular Sequence Data; Muscle, Skeletal; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Proteins; Transgenes; Transgenic animals and transgenic plants; Viral Proteins - genetics; Zebrafish - genetics; Vertebrata; Recombination; conditional transgenesis; transgenic fish; Copy number; Pisces; Green fluorescent protein; Transgenic animal; transgene copy number; green fluorescent protein (GFP); Transgenesis
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-004-5790-z

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp (green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.