Evaluation of a fish gene transfer system : expression, fate, and germline transmission of CAT recombinant plasmid and phage sequences microinjected into newly fertilized eggs of the Japanese medaka, Oryzias latipes (Temminck & Schlegel)

The creation of 'transgenic' animals has provided insights into mechanisms of gene regulation, as well as opened up a new avenue for genetic improvement of livestock, including fish. In this thesis, the suitability of the Japanese ricefield fish or 'medaka' (Oryzias latipes) as a gene expression system was evaluated. The procaryotic chloramphenicol acetyltransferase (CAT) gene regulated by a double eucaryotic promoter-enhancer region was chosen as a reporter. This reporter was introduced as either a supercoiled or linear recombinant plasmid (pUSVCAT), as a phage, or as purified phage DNA. DNA or phage was microinjected into the cytoplasm of newly fertilized medaka eggs at the 1-2 cell stage. Expression and fate of the injected DNA or phage were monitored by harvesting medaka at various developmental stages and performing CAT enzyme assays and Southern blot analyses, respectively. Several injected eggs were allowed to develop to sexual maturity, and their offspring were pooled and tested by CAT enzyme assay for inheritance of the CAT sequences. The patterns of expression of injected supercoiled and linear pUSVCAT DNA were very similar, indicating that DNA conformation does not affect the efficiency of expression. CAT enzyme activity was detectible from the early high blastula stage (4 hr post-injection), was strongest at the late gastrula/early neurula stage (1 day post-injection), and was sustained but slightly weaker in the one-week old embryo. Expression was significantly reduced in hatchlings (2 weeks post-injection), varying noticeably among the individuals analysed. CAT expression was still detectible in free-swimming fish (4 weeks post-injection). Recombinant CAT phage particles or purified CAT phage DNA were also able to express the CAT gene up to the free-swimming fish stage. However, in these treatments, the strongest CAT expression was seen in the one-week old embryo instead of in the gastrula/neurula, raising the possibility of a role played by different vector sequences on gene expression. Studies on the fate of injected supercoiled and linear pUSVCAT revealed conversion of the input forms to high molecular weight head-to-tail and randomly oriented concatemers respectively. Total plasmid DNA increased rapidly during cleavage and gastrulation, indicative of plasmid replication, whereas degradation of plasmid sequences was observed by the early high blastula stage. In the gastrula/neurula derived from injection of supercoiled pUSVCAT, total plasmid DNA increased ten-fold, whereas injection of linear pUSVCAT resulted in a 12-fold increase at the same stage. In both cases, most of the observed increase was contributed by the high molecular weight concatemers. The amount of plasmid DNA decreased after the gastrula/neurula stage, and this DNA was exclusively of the high molecular weight form at hatching and could persist to the free-swimming stage. Neither the DNA from injected CAT phage particles nor the injected purified CAT phage DNA appeared to be concatenated during early embryogenesis. In both cases, however, the phage DNA appeared as higher molecular weight DNA by the one-week old embryonic stage, probably formed by covalent end-to-end ligations. DNA of CAT phage particles did not increase until after the early high blastula stage, but by the flat blastula stage (10 hr post-injection) a three-fold increase over the input amount was observed. There was no significant increase at the gastrula/neurula stage, nor was there an immediate decrease thereafter. Injected purified CAT phage DNA increased through the stages of cleavage and gastrulation, the gastrula/neurula having seven-fold more CAT phage DNA than that injected, and decreased thereafter. Both DNA of injected phage particles and injected phage DNA could persist to the free-swimming stage. CAT gene expression was detected in a number of pooled offspring from several DNA and phage-treated fish, indicating inheritance of the input sequences. The data in this study suggest that the germline-positive parents are probably mosaic for the presence of the CAT sequences, and that germline transmission is possible with plasmid DNA of both conformations, DNA-carrying phage particles, or purified phage DNA. The above results, coupled with the ease of handling and manipulation of the medaka embryo, strongly favour the use of the medaka as a transient expression and transgenic animal model.Science, Faculty ofZoology, Department ofGraduat