PUBLICATION
FLPe functions in zebrafish embryos
- Authors
- Wong, A.C., Draper, B.W., and Van Eenennaam, A.L.
- ID
- ZDB-PUB-100621-27
- Date
- 2011
- Source
- Transgenic Research 20(2): 409-415 (Journal)
- Registered Authors
- Draper, Bruce, Van Eenennaam, Alison L., Wong, Andrew C.
- Keywords
- FLP, FLPe, Recombinase, Zebrafish
- MeSH Terms
-
- Animals
- Animals, Genetically Modified*
- DNA Nucleotidyltransferases/genetics
- DNA Nucleotidyltransferases/metabolism*
- Embryo, Nonmammalian/metabolism*
- Female
- Fertilization/genetics
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism
- Male
- Microinjections
- Mosaicism
- Recombination, Genetic
- Saccharomyces cerevisiae/genetics*
- Saccharomyces cerevisiae/metabolism
- Transgenes/physiology*
- Zebrafish/genetics
- Zebrafish/metabolism*
- PubMed
- 20552273 Full text @ Transgenic. Res.
Citation
Wong, A.C., Draper, B.W., and Van Eenennaam, A.L. (2011) FLPe functions in zebrafish embryos. Transgenic Research. 20(2):409-415.
Abstract
To assay the efficiency of the FLP/FRT site-specific recombination system in Danio rerio, a construct consisting of a muscle-specific promoter driving EGFP flanked by FRT sites was developed. FLPe capped RNA was microinjected into transgenic single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 h post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; P < 0.001) than would be expected from a cross in the absence of the recombinase (50%). Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR analysis and FLPe-mediated recombination was confirmed by DNA sequencing. Sperm derived from confirmed transgenic males in these experiments was used to fertilize wild-type eggs to determine whether germline excision of the transgene had occurred. Clutches sired by FLPe-microinjected males contained 0-4% fluorescent embryos. Transgenic males that were phenotypically wild-type produced no fluorescent progeny, demonstrating complete excision of the transgene from their germline. FLPe microinjected males that retained some fluorescent muscle expression produced a small proportion of fluorescent offspring, suggesting that in mosaic males not all germline cells had undergone FLPe-mediated transgene excision. Our results show that FLPe, which is derived from Saccharomyces cerevisiae, is an efficient recombinase in zebrafish maintained at 28.5 degrees C.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping