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

Abstract

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.

Evidence for a circaseptan and a circasemiseptan growth response to light/dark cycle shifts in nucleated and enucleated Acetabularia cells, respectively.

“…To (25) by microinjecting (26) Drosophila gene constructions that are known to affect circadian and ultradian rhythms (27)(28)(29)(30). The transformed progeny of such Acetabularia cells might then be used to determine whether a change in a circadian rhythm would also affect a circaseptan rhythm.…”

kurkku, a Phenotype of Acetabularia acetabulum That Is Arrested in Vegetative Growth, Can Be Rescued with Wild-Type Cytoplasm.

“…How a wild type compensates may well be mutant specific: kurkku and our 10 other developmentally arrested phenotypes are not identically compensated in wildtype grafts. Because A. acefabulum tolerates microinjection (nuclei; Neuhaus et al, 1986), it may be possible to distinguish those phenotypes compensated equally well by subcellular fractions as by intact apices from other phenotypes that can only be compensated by structurally intact apices.…”

“…Its giant size and ability to heal wounds (Menzel, 1980;Fester et al, 1993) have facilitated experimental manipulations, such as cell grafting, amputation, enucleation, and microinjection (Berger et al, 1987;Menzel, 1994), and have permitted the age or developmental potential of pieces of the cell and intact cells to be analyzed independently (Runft and Mandoli, 1995). In addition, the organism can be stably transformed (Neuhaus et al, 1986); it expresses and correctly targets proteins encoded by heterologous or foreign DNA (Neuhaus et al, 1984), and it is amenable to biochemical, "nucleocytoplasmic" (Berger et al, 1987), and genetic manipulations (Mandoli and Larsen, 1993). The stalk of the vegetative cell of A. acetabulum is decorated with rings, or whorls, of sterile hairs.…”

Advances in genetic engineering of marine algae

“…However, due to the difficulties of the immobilization of the algal cells, the complicated manipulation technology, and the low numbers of microinjected cells at a given time that can be achieved (Neuhaus and Spangenberg, 1990), the microinjection method is rarely used in marine algal transformation. A high yield and stable nuclear transformation was achieved in the marine unicellular green alga Acetabularia mediterranea by microinjecting SV40 DNA and pSV2neo into the isolated nuclei of algal cells and then implanting the injected nuclei into anucleate cell fragments of the same species (Neuhaus et al, 1986). This established and high yield method was successfully used to tackle the problem of the nuclear transport of algal proteins (Pfeiffer et al, 2009).…”

“…In the late 1980s, several eukaryotic marine microalgae and seaweeds were successfully transformed by different transformation methods, e.g., microinjection in the marine macro-green alga Acetabalaria sp. (Neuhaus et al, 1986), plasmid vectors in the marine diatom Cyclotella cryptica (Dunahay et al, 1995), and gene gun or the biolistic method in the macro-red alga Eucheuma sp. (Kurtzman and Cheney, 1991) and brown alga Laminaria japonica (Qin et al, 1998).…”