Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct.
Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish.
Rembold
Martina
M
Developmental Biology Unit, EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Lahiri
Kajori
K
Foulkes
Nicholas S
NS
Wittbrodt
Joachim
J
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009990:Oryzias / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D019076:Transgenes / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics*
D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific