Nuclear transformation of Volvox carteri
B Schiedlmeier, R Schmitt, W Müller, M M Kirk, H Gruber, W Mages, and D L Kirk
Lehrstuhl für Genetik, Universität Regensburg, Germany
Stable nuclear transformation of Volvox carteri was achieved using the cloned V. carteri nitA+ gene (which encodes nitrate reductase) to complement a nitA- mutation. Following bombardment of mutant cells with plasmid-coated gold particles, putative transformants able to utilize nitrate as a nitrogen source were recovered with an efficiency of approximately 2.5 x 10(5). DNA analysis indicated that the plasmid integrated into the genome, often in multiple copies, at sites other than the nitA locus. Cotransformants were recovered with a frequency of 40-80% when cells were cobombarded with a selected and an unselected marker. Thus, V. carteri becomes one of the simplest multicellular organisms that is accessible to detailed molecular studies of genes regulating cellular differentiation and morphogenesis.
Proc Natl Acad Sci U S A. 1994 May 24; 91(11): 5080–5084.
Volvox carteri is a multicellular organism with a complete division of labor between somatic and reproductive cells (1, 2). Genetic analysis (1-4) has led to the hypothesis that a small number of loci act to cause differentiation of these two cell types (5), and patterns of cell-type-specific gene expression in wild-type and mutant embryos are consistent with that hypothesis (6). However, detailed molecular analysis ofthese putative regulatory loci has awaited a method for transforming the organism with exogenous DNA. We repeatedly tried to transform V. carteri with various bacterial or plant selectable markers that were introduced by microinjection, electroporation, particle bombardment (7), UV-laser microbeam irradiation (8), agitation with glass beads (9), etc. As with the related unicellular alga, Chlamydomonas reinhardtii, reproducible transformation with heterologous selectable markers was not achieved, possibly because of an inability of these algae to express heterologous genes. Again in parallel with C. reinhardtii (9-12), success in transforming V. carteri has come with the availability of a homologous selectable marker: here we report use of the recently cloned nitrate reductase-encoding gene of V. carteri, nitA (13), to complement a nitA mutation.