Linda Benedict, Cooper, Richard K. | 5/19/2009 11:43:56 PM
When a gene from one species of plant or animal is inserted into the genome of another species, the resulting plant or animal is then transgenic. Methods for transferring genes have one thing in common: the plasmid vectors used for gene delivery rely on random integration (insertion) of the transgene into the recipient chromosome. The result is usually an integration rate of 3 percent to 10 percent. With fish, it is usually 3 percent or less.
As we began to use transgenic techniques to investigate the possibility of enhancing disease resistance in channel catfish, it became apparent that a better method for obtaining stable integration was needed. Our goal has been to develop channel catfish resistant to disease by delivering to the catfish genome, as efficiently as possible, a gene encoding a lytic peptide. To circumvent the integration problems with previous transgenes, we designed a DNA vector (plasmid) that would force the incorporation of the desired gene into the targeted host genome.
Using a piece of mobile DNA called a transposon, we engineered a plasmid containing the lytic peptide within a transposon. A transposon can be cut or removed from the vector containing it (in this case a plasmid) by an enzyme called a transposase. By controlling the transposase with a promoter (on/ off switch), we can regulate when the transposase removes the transposon from the plasmid and inserts it into another segment of DNA, that is, a catfish chromosome.
Once the transposon has been removed from the plasmid and inserted into the chromosome, the remaining plasmid DNA (containing the transposase gene) is destroyed. This removes the source of the transposase and eliminates the chance of the transposon being mobile in the future. The result is an efficient delivery system that creates stable DNA insertions with traits to be passed to the offspring. Work in our laboratory has resulted in more than than 50 percent transformation efficiencies in channel catfish, and the transgene has been stable in these fish for more than three years.
Similar results have been obtained in koi carp. To demonstrate the ability of the gene to be passed from parent to offspring, we injected male koi carp in the region of the testes. Two weeks after injection, sperm were harvested and used to fertilize eggs. The offspring were assayed four times during a year for the presence of the transgene. Sixty-six percent of the offspring were positive after one year, and sperm from three of the five males remained positive for the transgene after one year. This vector is a useful tool for delivering a desired gene to the genome of a wide array of animals and possibly plants. In addition to its value inproducing transgenic fish, the vector has potential for being used in gene therapy applications to generate large numbers of cells with a transgene stably incorporated in to their genome.