High yield and stable transformation of the unicellular green alga Acetabularia by microinjection of SV40 DNA and pSV2neo

SV40 DNA and pSV2neo were microinjected into isolated nuclei of Acetabularia mediterranea. The injected nuclei were implanted into anucleate cell fragments of the same species. Such combinations not only survived but also formed progeny. The F₁, F₂ and F₃ generations of these combinations were analyzed. In the case of SV40-treated cells T-antigen was expressed and accumulated in the nuclei of all three generations studied as shown by indirect immunofluorescence. Nuclear exchange experiments revealed expression of the T-antigen only if a transformed nucleus but not if only a transformed cytoplasm was involved. Transformation by pSV2neo, a chimeric gene with a selectable marker was demonstrated by the induction of G-418 resistance as well as immunofluorescence. Genomic DNA was isolated from gametes, originating in cysts from the F₁, F₂ and F₃ generations of injected cells, and subjected to Southern analysis. These experiments demonstrated that both types of DNA are integrated into the host genome.