Sequences of transgene insertion sites in transgenic F4 common carp

In our previous study, fast-growing transgenic common carp (Cyprinus carpio) were produced by microinjection of BamHI-digested plasmid pMThGH into common carp fertilized eggs (Zhu et al., 1989). To keep the genetic diversity of transgenic group, we produced the transgenic F1 offspring by crossing one transgenic female with four transgenic males. The transgenic F4 common carp were raised by hybridization between the transgenic male and transgenic female generation by generation. In F4 generation, 100% of the offspring carried pMThGH-transgene and the transgenic offspring had improved growth rate and feed utilization efficiency compared with the controls (Fu et al., 1998). However, up to the present, integration information of the transgene in transgenic F4 fish has been lacking. Since firstly described by Perucho et al. (1980), the technique of plasmid rescue has been utilized in the study of various transgenic species, but it has not, to our knowledge, been employed previously in the study of transgenic fish. In this study, by use of the technique of plasmid rescue, the sequences of transgene insertion sites in transgenic fish were characterized in two heaviest individuals of 1-year-old transgenic F4 fish. Total DNAs were partially digested with BamHI, gel recovered, and treated with T4 DNA ligase. The self-ligated DNA was transformed into E. Coli, and ampicillin-resistant clones were selected for plasmid preparation and analysis. Among the recovered plasmids, in spite of those having the same configuration as the original pMThGH, the other five types have aberrant configurations, which were quite different from the original configuration. These aberrant plasmids were