Extrachromosomal plasmid DNA transmission and amplification in Metaseiulus occidentalis (Acari: Phytoseiidae) transformants generated by maternal microinjection

Abstract

The plasmid pJKP2 912.5 kb) was injected into Metaseiulus occidentalis females using a maternal microinjection procedure (Presnail and Hoy 1992). DNA transmission to their progenies was followed by the polymerase chain reaction (PCR) for several generations. After selecting and retaining individuals that tested positive by the PCR for 5 generations, individual females were used to initiate 12 separate transgenic lines (T1-T12). Southern blot analysis performed 10 generations after injection indicated that in at least 8 lines the plasmid DNA was transmitted extrachromosomally, and in 5 of these lines the extrachromosomal plasmid DNA was highly amplified with an estimated 10 million copes per female. The longevity, fecundity, immature mortality, and sex ratio of these transgenic lines were not different from the wild-type control under laboratory conditions. However, after 20 generations, few transformants tested positive by the PCR and Southern blot analysis indicated no distinct banding pattern. Results from Southern blot analysis and the PCR indicated the extrachromosomal plasmid DNA was completely lost after 30 generations, suggesting a fitness cost did occur. Maternal microinjection delivers DNA efficiently to M. occidentalis and could be used for testing selectable markers or to deliver transposable-element vectors to obtain high rates of chromosomal integrations.