Isolation and characterization of Tilapia (Oreochromis mossambicus) insulin-like growth factors gene and proximal promoter region

To understand the molecular mechanism which controls the transcription of the insulin-like growth factors (IGFs) gene, we have cloned and sequenced the cDNA for the proximal promoter region of the tilapia IGFs gene and have characterized its activity by chloramphenicol acetyltransferase (CAT) transient transfected expression assays. Tilapia (Oreochromis mossambicus) IGF-1 cDNA (549 bp) was amplified by PCR from single-stranded cDNA of growth hormone (GH)-induced liver RNA using a pair of oligonucleotides specific for fish IGF-I as amplification primers. Tilapia IGF-I and IGF-II 5' termini were analyzed by rapid amplification of cDNA 5' ends (5'RACE), Analysis of the 5'RACE results revealed two transcription start sites in IGF-I and one transcription start site in IGF-II. Different fragments of the 5' flanking region were transfected into human lung adenocarcinoma cells. In the cell line, maximum promoter activity was located in the distal 657 basepairs of the IGF-I 5' flanking region and in the distal 450 basepairs of the IGF-II 5' flanking region. The in vivo actions of the IGFs promoter on developmental stage expression were investigated further in transgenic zebrafish in which an IGFs promoter-driven green fluorescent protein (GFP) encoding the cDNA transgene was microinjected into embryos. Morphologic and RT-PCR studies of the transgenic zebrafish indicated that IGF-I promoter-driven GFP transcripts appeared for the first time in the l-K-cell stage and the IGF-II promoter-driven GFP transcripts appeared for the first time in the 32-cell stage. Fluorescent (GFP) distribution was apparent within 48 h in IGF-II-transgenic zebrafish embryos, especially in eye, muscle, corpuscle, floor plate, horizontal myoseptum, yolk sac extension, and yolk sac. These results indicate that the IGF-I and IGF-II promoters are active in tissue and in a development-specific manner. Our findings also indicate that the IGF-II promoter influences the growth of fish embryos earlier than does IGF-I, and IGF-II has higher levels of expression than does IGF-I. These results suggest that the IGF-II promoter plays a growth factor role in teleost embryo development.