Analysis of cell-specificity and variegation of transgene expression driven by salmon prolactin promoter in stable lines of transgenic rainbow trout

Abstract

In order to identify the specificity and functionality of salmon prolactin (sPRL) promoter, transgenic rainbow trout carrying a construct comprising the 2.4 kb fragment of the 5′ flanking region of Atlantic Chinook sPRL gene fused either to the reporter genes cat (sPRL-cat) or lacZ (sPRL-lacZ) were produced, sPRL-cat in transgenic F0 fish expressed strongly CAT only in the pituitary gland. Transgenic in F1-F4 lines harbouring sPRL-lacZ expressed β-galactosidase (β-gal) only in the follicular PRL-producing cells of the adenohypophysis. We observed heterocellular, mosaic distribution of β-gal within PRL cell population and enormous variation of lacZ expression level between the littermates in the same transgenic line. Regardless of the transgene copy number, age or sex of transgenic fish, β-gal expression was lactotroph-specific but variegated in all the nine F2 hemizygous lines analysed. One line harbouring a multicopy integration was followed up to F4 generation: the transgene was transmitted without modifications. Analysis of genomic DNA from pituitaries showed that lacZ sequences were highly methylated. LacZ expression was low and its transcripts, analysed by in situ hybridisation, showed a mosaic distribution within the pituitary gland. These data suggest that variegated expression of lacZ can occur at the transcription level owing to the silencing effect of lacZ gene. After proving the tissue-specific expression of reporter genes driven by the sPRL promoter, we tried to obtain the genetic ablation of PRL-producing cells, by transferring the same construct comprising diphtheria toxin DT-A gene (tox). However, the high mortality rate of sPRL-tox transformed embryos has embedded this study and no transgenic fish expressing tox were produced. The appropriateness of using transgenic strategies to analyse gene function in Salmonids is discussed, especially the implications of the multicopy integration patterns and of the variegated transgene expression.