Introducing foreign genes into fish eggs with electroporated sperm as a carrier.
A new method has been developed for introduction of foreign genes into fish eggs. The procedure is based on the incubation of fish sperm cells suspended in dilute citrate solution with plasmid DNA, followed by application of high-field-strength electrical pulses (electroporation) to increase DNA binding., uptake, or both. Tissue homogenates and genomic DNA extracts of free swimming fry developed from eggs fertilized with treated sperm was tested to evaluate the efficiency of gene transfer. Dot blot hybridization and gene expression assay demonstrated the presence and expression of the reporter genes introduced in 2.6 to 4.2% of several hundreds of tested larvae of common carp (Cyprinus carpio L.), African catfish (Clarias gariepinus), and tilapia (Oreochromis niloticus). No transgene has been found in the fry resulting from parallel experiments without sperm electroporation. This is the first report on successful application of electroporated sperm cells for production of transgenic fish.
Müller
F
F
Institute for Molecular Genetics, Agricultural Biotechnology Center, Gödöllö, Hungary.
Ivics
Z
Z
Erdélyi
F
F
Papp
T
T
Váradi
L
L
Horváth
L
L
Maclean
N
N
Orbán
L
L
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D002347:Carps / Q000235:genetics; D002397:Catfishes / Q000235:genetics; D003584:Cytological Techniques; D004247:DNA / Q000032:analysis; D005260:Female; D005306:Fertilization; D005399:Fishes / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008297:Male; D010063:Ovum / Q000502:physiology; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*; D017210:Tilapia / Q000235:genetics; D014162:Transfection / Q000379:methods*
D004247:DNA