GENERATION OF CHIMERIC FISH EMBRYO BY CELL TRANSPLANTATION

Abstract

Cell mediated gene transfer systems allow transgene to be inserted into host genomes at precise loci through homologous recombination. This method has tremendous application in transgenic animal production, especially in valuable species that expressed pharmaceutical proteins. To develop method for performing insertional transgene in fish by cell-mediated' transfer, we introduced cells isolated from transgenic carp embryos into non-transgenic recipients at blastula stage. Donor embryos used for cell isolation were obtained from transgenic carp (Cyprinus carpio) for human growth hormone. Cells were dissociated from embryos at 16 - 32 cell stage by trypsin digestion. Recipient embryos used for cell transplantation experiment were obtained from 000- transgenic carp. The recipient embryos at blastula stage were dechorionated with trypsin. Cell transplants were performed with aid of a micromanipulator. Approximately 20 - 100 cells were delivered into cell mass of each recipient blastula by micro injection. The survival embryos at stage 13 (after one day incubation) were examined for the presence of human growth hormone gene by PCR. In experimental group, the specific band for human growth hormone gene was detected, while in the control it was not observed. The results showed that cell from transgenic embryos at 16 - 32 cell stage can contribute to the germ line after transplantation to recipient embryos at blastula stage.