Position-independent expression and germline-transmission of transgenes in zebrafish (chromatin, gene expression)

Transgenic animals are useful for studying the regulation of gene expression during development. However, the expression of transgenes depends on the chromosomal environment at the sites of integration. The variability in expression of a transgene, which is the consequence of influences of surrounding chromatin at the sites of integration, is referred to as a "position effect". Border elements are DNA sequences that can alleviate position effects in transgenic animals. Zebrafish, Danio rerio, is an excellent model system for studying vertebrate development. Transgenes are subject to position effects in zebrafish. The goal of this study was to examine the ability of two types of border elements, the scs/scs$spprime$ from the D. melanogaster 87A7 heat shock locus and the A-element from the chicken lysozyme gene, to protect transgenes from position effects in zebrafish. The transgenic construct used in this study, FV3CAT, consisted of the transcriptional regulatory region from the carp $beta$-actin gene, the chloramphenicolacetyltransferase (CAT) gene and the $3spprime$-untranslated region from the chinook salmon growth hormone gene. The expression patterns of FV3CAT in zebrafish embryos were examined in transient expression assays. FV3CAT was expressed in all tissues of developing embryos. The abilities of scs/scs$spprime$- and A-elements to alter expression levels of the FV3CAT were tested in transient assays. Neither scs/scs$spprime$- nor A-elements altered expression levels of the FV3CAT, suggesting that neither border element functioned as enhancer and/or silencer in zebrafish. FV3CAT alone, or flanked by either scs/scs$spprime$-elements or A-elements, was introduced into zebrafish chromosomes and the patterns of transgene-expression were examined in multiple generations of zebrafish. The scs/scs$spprime$-elements were able to provide uniform and copy number-dependent expression of the FV3CAT in two generations of transgenic zebrafish. A-elements were able to protect the FV3CAT from influences of neighboring chromatin in three generations of zebrafish. Zebrafish which harbor transgenes flanked by either scs/scs$spprime$- or A-elements could be used for comparing mechanisms of function of these two types of border elements. Additionally, position-independent expression of transgenes flanked by scs/scs$spprime$-elements will allow the use of transgenic zebrafish for studying regulation of gene expression during vertebrate development.