In vitro and in vivo gene transfer in rainbow trout, Oncorhynchus mykiss.

摘要

Studies were conducted to examine the regulation of gene expression and modes of entry of recombinant DNA vectors in rainbow trout (Oncorhynchus mykiss) in vitro and in vivo. The bovine growth hormone genomic gene and cDNA were used as reporter genes in a number of plasmids containing a variety of mammalian and piscine promoter elements. Additionally, the chum salmon (Oncorhynchus keta) growth hormone genomic gene was cloned, sequenced, and tested for expression in one mammalian and four fish cell lines.;Transient expression assays in vitro showed the mammalian cell line (mouse L cells) was able to utilize several mammalian and one piscine promoters to drive expression of both forms of the bovine growth hormone genes. Metallothionein-derived promoters were inducible with zinc in a dose-dependent manner. Expression of the chum salmon growth hormone gene, driven by a strong mammalian promoter, was low compared to expression of the mammalian genes in a mammalian cell line.;In contrast, none of the fish cell lines examined were able to express measurable quantities of any growth hormone gene products. Further analysis revealed a transcriptional processing problem of the mammalian gene by the piscine cells.;Microinjection of foreign DNA into rainbow trout eggs was accomplished by two methods. Compared to non-injected control eggs, both microinjection techniques significantly impacted fish survival through hatching. Genomic analysis of trout fry revealed the presence of the foreign DNA in 14% at 55 days post-fertilization.;This study indicates that selection and origin of genes and regulatory elements must be carefully considered and tested before use in transgenic fish production. Analysis of gene expression in vitro is an important first step in determining a recombinant DNA vector's usefulness.