Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system.
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.
Pan
Xiufang
X
Department of Biological Sciences, National University of Singapore, Singapore.
Wan
Haiyan
H
Chia
Wendy
W
Tong
Yan
Y
Gong
Zhiyuan
Z
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D005786:Gene Expression Regulation; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics*
D005819:Genetic Markers; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases