Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs.
Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.
Müller
F
F
Institute for Molecular Genetics, Agricultural Biotechnology Center, Gödöllô, Hungary.
Lele
Z
Z
Váradi
L
L
Menczel
L
L
Orbán
L
L
D000818:Animals; D030801:Animals, Genetically Modified; D002347:Carps; D002397:Catfishes; D004558:Electric Stimulation; D004625:Embryo, Nonmammalian / Q000502:physiology; D005260:Female; D005306:Fertilization; D005399:Fishes; D015870:Gene Expression; D008156:Luciferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008297:Male; D010063:Ovum / Q000502:physiology*; D010902:Pituitary Gland / Q000502:physiology; D010957:Plasmids; D013094:Spermatozoa / Q000502:physiology; D014162:Transfection; D015027:Zebrafish; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D008156:Luciferases; D001616:beta-Galactosidase