Electroporation: a method for transferring genes into the gametes of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio).
Recombinant plasmids containing the Rous sarcoma virus long-terminal repeat (RSVLTR) promoter linked to either rainbow trout (Oncorhyncus mykiss) growth hormone 1 (rtGH1) or growth hormone 2 (rtGH2) cDNA were linearized and introduced into the fertilized eggs of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio) by both electroporation and microinjection. The latter two species had these rainbow trout constructs (RSVLTR-rtGH1cDNA or RSVLTR-rtGH2) electroporated into both gametes (i.e., sperm and unfertilized eggs) prior to fertilization, into eggs shortly after fertilization, and at the first cell division stage. Survival was determined just after hatching and again between 3 and 5 months after hatching. Polymerase chain reactions and Southern blot analyses were used to detect those individuals carrying the introduced foreign genes 3 to 5 months after hatching, respectively. Individuals analyzed by both methods yielded identical results in a double-blind study. The electroporation results were compared with groups that were microinjected. Although survival was similar, electroporation tended to produce a greater number of transgenic individuals than the microinjection procedure, and many more eggs could be treated per unit time by electroporation than microinjection. Survival was better for common carp when electroporation was performed shortly after fertilization, whereas channel catfish fared better at the first cell division stage. Electroporation prior to and shortly after fertilization, and at the first cell stage appeared to generate a large fraction of transgenic fish. We cautiously conclude that electroporation is an efficient method for introducing foreign DNA into fish gametes and embryos and may be an ideal method for treating large numbers of gametes in a modest period.
Powers
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Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950.
Hereford
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Cole
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Chen
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Lin
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Kight
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Creech
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Dunham
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R
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002347:Carps / Q000235:genetics*; D003584:Cytological Techniques; D005260:Female; D013006:Growth Hormone / Q000235:genetics; D007059:Ictaluridae / Q000235:genetics*; D008297:Male; D008845:Microinjections; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods*; D015027:Zebrafish / Q000235:genetics*
D013006:Growth Hormone