Development of vectors for expression of transgenes in fish: Regulation of the carp beta-acting gene

Fish are excellent model systems for biological research. Numerous, large, transparent, and externally fertilized eggs of many species make them most suitable for genetic manipulation, especially for production of transgenic animals. Research on genetic engineering of fish requires suitable expression vectors, in which strong genetic regulatory elements are required to drive transcription. Accordingly, I have cloned and sequenced the carp beta-actin gene, identified its regulatory elements, and developed two fish expression vectors using the identified regulatory elements.;The CAAT and TATA boxes were required for the promoter activity. The CArG box between the CAAT and TATA boxes was not required for simple promoter activity in tissue cultured cells, but was required in conjunction with a second CArG box in the first intron to give full promoter activity in dividing cells of growing embryos. Mobility shift experiments showed that either the same or similar protein factor(s) bind to both the proximal promoter and to the CArG region in the first intron. Lambda exonuclease mapping experiments indicated that protein factor(s) bind around the CArG boxes. These results suggest that interactions between the proximal promoter and the first intron are involved in the regulation of the carp beta-actin gene.;Using the information gained from the above experiments, two fish expression vectors, FV-1 and FV-2, were developed. They contain the proximal promoter and enhancer regulatory elements of the carp beta-actin gene and the polyadenylation signal from the salmon growth hormone gene. The two fish expression vectors have been tested in tissue cultured cells and transgenic fish. Gene expression from these vectors was higher than the other widely used expression vectors. FV-1 and FV-2 should be useful for genetic engineering of fish.;The carp beta-actin gene consists of six exons and spans 3.6 kb. Several regulatory elements of the gene have been identified. These include the proximal promoter, an upstream negative regulatory element, and positive and negative regulatory elements in the first intron. The proximal promoter consists of typical CAAT and TATA boxes and an evolutionarily conserved sequence CC(A/T)...