Isolation and characterisation of tilapia ß-actin promoter and comparison of its activity with carp ß-actin promoter
Abstract
The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the ß-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a ß-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia ß-actin regulatory sequence, (2) 1.5 kb carp ß-actin regulatory sequence, and (3) 4.7 kb carp ß-actin regulatory sequence. Although the 1.6 kb tilapia ß-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp ß-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp ß-actin regulatory sequence, the 1.6 kb tilapia ß-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia ß-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp ß-actin or the 1.5 kb carp ß-actin regulatory sequences. The 1.6 kb tilapia ß-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp ß-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia ß-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.
Citation
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression (2003) 1625 (1) 11-18 [doi:10.1016/S0167-4781(02)00534-1]