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The optimal transfection conditions for efficient transgene delivery into a specific cell type should be empirically determined, particularly in cases involving unusual cell types. We compared the conditions for effective introduction of transgenes into Siberian sturgeon (Acipenser baerii) cell lines by evaluating the cytotoxicity and transfection efficiency of three commercially available transfection reagents: Lipofectamine 2000, X-tremeGENE HP DNA Transfection Reagent, and GeneJuice Transfection Reagent. Plasmid vectors containing the gene encoding enhanced green fluorescent protein were mixed with each of the transfection reagents using reagent-to-plasmid ratios of 1:1, 2:1, and 4:1. Then, the complexes were used to transfect three Siberian sturgeon cell lines derived from the heart, head kidney, and gonad. Cytotoxicity and transfection efficiency were measured via flow cytometry after propidium iodide staining. No significant cytotoxicity was observed at the optimal treatment conditions in all cases, with the exception of Lipofectamine 2000-treated gonad-derived cells. Although the transfection efficiencies in A. baerii cells were generally low, X-tremeGENE HP DNA Transfection Reagent showed the highest transfection efficiency at ratios of 2:1 or 4:1, depending on the cell type. Hence, X-tremeGENE HP DNA Transfection Reagent can be used to effectively transfer foreign genes into three A. baerii cell lines.

This study was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture. Total 48 trials of culture employing low and middle cell densities of less than 5.5 × 10(4) cells/cm(2) in initial seeding did not induce cell survivals (0%, 0/48), but the trials in high cell density of more than 5.5 × 10(5) cells/cm(2) could induce cell survival and primary cell attachment on the plate (88.9%, 24 in 27 trials). When all initially attached cell populations were continuously cultured in two different media, only five cell populations that were enzymatically isolated and cultured under Leibovitz's L-15 medium could grow up to more than 40th subculture. Each cell population was stably cultured according to its own growth rate and all showed normal diploid DNA contents. Two morphologically different cell types that has an elongated shape or a round shape were identified in culture, which was subsequently identified that two cell types are considered as a fibroblast (an elongated shape) and a vascular endothelial cell (a round shape) on the basis of the results of gene and protein expression analysis. Additionally, the sufficient number of viable cells could be successfully retrieved after freezing and thawing from all five cell populations suggesting the feasibility of long-term cryopreservation of the cells. The data and cells obtained from this study will contribute to development of in vitro model for basic biological studies using sturgeon species.

To develop combined vaccine systems, we have generated Edwardsiella tarda ghosts (ETG) displaying a foreign protein on the outer membrane and also Ed. tarda cadaver (ETC) expressing a heterologous protein in the cytoplasm. Green fluorescent protein (GFP) was used as a model foreign protein. A constitutive promoter (EtPR C28-1) cloned newly from Ed. tarda was used as a promoter for the expression of foreign protein. Comparison of the strength of the new promoter with a commercially available constitutive promoter (P(HCE)) showed higher expression levels of the novel expression system. The N-terminal domain of ice nucleation protein (InaN), an outer membrane protein of Pseudomonas syringae, was used as an anchor motif for surface display of GFP. By transformation of Ed. tarda with the constructed vectors, GFP was successfully expressed on the surface of ETG and in the cytoplasm of ETC. When compared to P(HCE) driven expression, approximately more than 2 times of GFP was expressed on ETG and in ETC by EtPR C28-1 promoter when judged by fluorescent spectrophotometry. Furthermore, significantly higher expression of GFP on the surface of ETG by EtPR C28-1 than by P(HCE) was demonstrated by serum agglutination assay. These results suggest that the newly cloned Ed. tarda constitutive promoter is capable to express foreign proteins not only on the surface of Ed. tarda ghosts but also in the cytoplasm of Ed. tarda cadavers, and can be used as an efficient promoter for the expression of heterologous antigens of the ETG and ETC-based combined vaccines.

Vibrio anguillarum ghosts (VAG) were generated, for the first time, using a conjugation vector containing a ghost bacteria inducing cassette, pRK-lambdaP(R)-cI-Elysis, in which the expression of PhiX174 lysis gene E was controlled by the P ( R )/cI regulatory system of lambda phage. By scanning electron microscopy, holes ranging 80-200 nm in diameter were observed in the VAG. To avoid the presence of bacterial genomic DNA and an antibiotic resistance gene in the final VAG product, we constructed a new dual vector, pRK-lambdaP(R)-cI-E-SNA, containing the E-mediated lysis cassette and the staphylococcal nuclease A (SNA)-mediated DNA degradation cassette, and generated safety-enhanced VAG for use as a fish vaccine.