From a number of commercially available DNA carriers we decided to try a synthetic cationic lipopolyamine molecule already tested succesfully in cultured eukaryotic cells with a neglectable degree of toxicity and without interfering with normal physiological processes. The reagent, commercially known as Transfectam (PROMEGA USA), interacts strongly with the DNA converting it into a DNA-carrier complex which mimics a cationic lipid layer. This transient structure readily associates with the target cell membrane through a cooperative ionic type interaction, which in turn promotes selective DNA uptaking and its subsequent endocitosis.

We have optimized conditions to transfect the fish cell line CHSE-214 to measure expression, maintenance and putative chromosomal integration of the reporter gene LUC, in the two versions of the bacterial plasmids shown in Figure 1.

First we compared two mechanisms to introduce the foreign DNA into the fish genome: DEAE-dextran, a normal reagent mammalian cell transfection, and Transfectam, a gentle and commercially available lipopolyamine carrier.

Transfection experiments (the act of DNA uptake by cells) were carried out with 1.0-5.0 m g plasmid DNA in a final volume of 500 m l of serum-deprived MEM on the fish cell line CHSE-214.

A second set of experiments were carried out to define the minimal amount of plasmid DNA as well as of the transfection reagents to avoid cell toxicity.

Once achieved, we set a defined time-span to evaluate persistence and localization of the foreign DNA inside the host cells by means of Southern blot analysis

Finally, we measured the expression of the foreign DNA in the host cells.

Transfectam turned out to be an adequate carrier to introduce DNA into cultured fish cells. It also allowed us to set the basic experimental conditions to be used in the remaining assays reported in this communication.

For maintenance experiments, two sets of four 75 cm² bottles were seeded each time for each vector and one was processed and the remaining bottles were incubated for 96 h.. In this time, one bottle was tripsynized, diluted and divided into two new ones for DNA extraction and the other used as a source for new generations. 5 generations were analized, and cell proccessed 30 min. post-transfection, bottle transfected without divide and cell untransfected were used as controls. Both plasmids were consistently detected after five succesive passages, considering that each passage involves cell dilutions. This is a persuasive indication that both plasmids can replicate in a highly different cellular environment when compared to their original bacterial host. Moreover, the addition of the fish repetitive sequences (SmaI) seems to increase its survival in transfected cells, which might mean that a putative genomic integration of the selected sequences could be taking place.

The results of 15 independent measurements expression in crude cell extracts, clearly demonstrated that incorporated DNA retains its full potential for expression. Using a standard calibration curve constructed with triplicate dilutions of commercial luciferase, we estimated that in our assays we detect in the order of 150 molecules per cell, suggesting that on the average, most transfected cells seem to retain the target enzymatic activity.

A minimal ratio of one to one, lipopolyamine carrier to plasmid DNA, was enough to efficiently transfect the cell line to follow the fate of target DNAs up to five cell passages. In this time-span we demonstrate the maintenance of the foreign DNA in the cells, the concomitant expression of the reporter gene, and a higher stability in time of p103 over the control plasmid which suggest a higher potential for integration. Thus, in this report we define an efficient model system for future "in vitro" evaluation of potential target genes of commercial interest for fish transgenesis.

The experiments reported here also provide a suitable "in vitro" system to approach pending basic questions relevant to commercial applications of transgenesis in fish. These include transient expression, putative genomic integration, and mosaicism of selected target genes.