Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

Abstract

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Transgene constructs in coho salmon (Oncorhynchus kisutch) are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-transmitted parasite DNA

“…Where bacterial plasmids have not been removed from construct sequences prior to injection, partial integrated transgene structures have been be assessed by plasmid rescue techniques. For example, in the case of a transgenic carp containing approximately 200 copies of a human GH gene construct at 4-5 separate loci, construct sequences were found to be organized in a head-to-tail fashion and inserts were associated with functional genes in the carp genome (Wu et al 2004(Wu et al , 2005, but the complexity of the strain and plasmid rescue technique precluded determination of the structure of individual insertion sites. Some transgenic lines of fish contain multiple construct copies of modified host sequences located at a single integration site, and lack plasmid sequences (e.g.…”

Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos

“…In an effort to develop effective methods for site-directed integration and enhance the efficiency of stable transgene integration, researchers have analyzed transgene flanking sequences to identify potential integration hotspots 1,2. However, studies on transgene integration in mammals have shown that integration appears to be a random process and, although sequences in integration sites have some common structural features, no so-called integration hotspots exist.…”

Integration mechanisms of transgenes and population fitness of GH transgenic fish

“…The exogenous genes tend to form long-fragment concatamers. Southern blot analysis indicated that the transgenes were organized in head-to-tail, head-to-head or tailto-tail concatemers in the genome of the host fish [3,9,10]. The high copy numbers of transgene concatemers might exist as heterochromatin in the host genome, which would be subject to gene silencing [11,12].…”