Chapter 73 Transformation of Chlamydomonas reinhardtii

Transformation of Chlamydomonas reinhardtii is essential for confirming the relationship between DNA clones and genetic loci identified by mutation, and is very useful in generating tagged mutants for cloning genes of interest. The development of reliable procedures for such nuclear transformation of C. reinhardtii has led to greater usefulness of the organism in molecular studies of flagellar assembly and function. Exogenous DNA is stably integrated into the C. reinhardtii genome, although this integration is usually associated with rearrangements of transformed DNA, as well as of the host DNA. Multiple copies of the transgenes can be integrated, either at separate sites or in linked arrays. The most common problem in making transformation work with C. reinhardtii is the variability in transformation efficiency between strains. The best remedy for a poor strain is to cross it to wild-type strains, for example, strains 137c or 21gr and use the progeny of the mating for further transformations. Cell-wall-less strains, that is, cw15 are generally used to eliminate the need for autolysin treatment to remove cell walls, but it have been found that an incubation of nitrogen-starved cells under crowded conditions removes the cell walls efficiently. For arg^- strains, however, autolysin treatment is necessary for highest transformation efficiencies.