Simple and efficient transgenesis with meganuclease constructs in zebrafish.
In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, low transgenesis frequency), making the generation of transgenic lines a laborious task. Coinjection of I-SceI meganuclease with a reporter construct flanked by I-SceI sites overcomes these problems by earlier transgene integration into the host genome. Here, we provide an optimized protocol for I-SceI meganuclease-mediated transgenesis in zebrafish. This simple protocol provides a reliable method to transiently test tissue-specific reporter expression of meganuclease constructs in injected embryos (F0). Furthermore, it substantially facilitates the generation of multiple stable transgenic lines increasing transgenesis frequencies up to 45%, compared with 5% without I-SceI. The reliable reporter activity in F0 and the improved transgenesis frequency make this protocol a powerful tool for use in gain- and loss-of-function, cell tracing, and cell labeling experiments.
Soroldoni
Daniele
D
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Pfotenhauerstrasse 108, 01307, Dresden, Germany. soroldon@mpi-cbg.de
Hogan
Benjamin M
BM
Oates
Andrew C
AC
D000818:Animals; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D018095:Germ-Line Mutation; D049452:Green Fluorescent Proteins; D008297:Male; D008845:Microinjections; D029701:Saccharomyces cerevisiae Proteins / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific