Efficient gene transfer into Xiphophorus muscle and melanoma by injection of supercoiled plasmid DNA.
Muscle and melanoma tissue of fish in the genus Xiphophorus were examined for their ability to take up and express foreign DNA. Supercoiled plasmid DNA containing a firefly luciferase reporter gene with expression driven by the cytomegalovirus enhancer and thymidylate kinase promoter was directly injected into the muscle or melanoma of individual Xiphophorus. Expression levels gradually increased to a maximum at 6 days after injection in both tissues, and this level was maintained for at least 10 days after injection. In both muscle and melanoma, there was a clear relationship between dose injected and reporter gene activity, with maximal expression at a dose of 20 microg of plasmid injected. At higher doses expression levels declined, suggesting the possibility that the uptake mechanism can be inhibited by high concentrations of DNA. Histochemical localization using a beta-galactosidase construct revealed high expression of the enzyme in isolated muscle fibers. The activity of a second coinjected reporter gene, sea pansy (Renilla reniformis) luciferase, was highly correlated with the activity of the firefly luciferase reporter gene in both tissues (R2 >.940), suggesting that the majority of variation between samples results from variation in overall DNA uptake between individuals. When firefly luciferase activity is expressed as a function of activity of the coinjected reporter, the variation between samples is greatly reduced. As a result, small differences in activity between constructs can be detected. This demonstrates the usefulness of the system for gene expression analysis in vivo.
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Hopkins Marine Station, Stanford University, Pacific Grove, California 93950, USA.
Powers
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D000818:Animals; D003063:Cnidaria; D003532:Cyprinodontiformes; D004278:DNA, Superhelical; D005393:Fish Diseases / Q000378:metabolism / Q000473:pathology*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008545:Melanoma / Q000378:metabolism / Q000473:pathology / Q000662:veterinary*; D018482:Muscle, Skeletal / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D004278:DNA, Superhelical; D011994:Recombinant Proteins; D008156:Luciferases; D001616:beta-Galactosidase