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.

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Fish transgene expression by direct injection into fish muscle.

The ability of a promoter sequence to drive expression of a reporter gene can be determined by direct injection of copies of the cloned sequence into fish muscle, followed by biopsy of muscle from the site of injection. We describe a set of experiments in which copies of the constructs FV1 and FV2, both comprising a carp beta-actin promoter sequence spliced to the bacterial reporter gene CAT, were injected into the muscle of tilapia fish )Oreochromis niloticus) of between 5 and 8 cm body length. The site of injection was carefully determined so that biopsy samples could be recovered from the injection site 24 hours, 48 hours, and 7 days after injection. Biopsy samples of muscle were homogenized and used for CAT assays. CAT activity was successfully detected in many of the muscle samples.

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PubMedDate not supplied

Transgene transmission and expression in rainbow trout and tilapia.

We describe the production of transgenic rainbow trout (Oncorhynchus mykiss) and tilapia (Oreochromis niloticus) by microneedle injection of fertilized eggs with cloned copies of novel gene constructs. Two aspects of the work with transgenic trout are presented; namely, patterns of inheritance of transgenes by the F1 generation following in vitro fertilization of gametes from transgenic and nontransgenic fish, and the degree of DNA methylation of transgenes in different fish and in different tissues of the same fish. Work with transgenic tilapia has been only of short duration, and evidence is presented simply to indicate their transgenic status. Transient expression studies using the bacterial gene chloramphenicol acetyltransferase when driven by fish-derived promoters are also discussed.

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