Nuclear Transformation: Technology and Applications

Abstract

SummaryNuclear transformation is now simple and efficient, and it has revolutionized the kinds of questions that can be addressed using Chlamydomonas reinhardtii as a model system. The highest rates of transformation are obtained using C. reinhardtii genes that complement auxotrophic mutations as selectable markers. Cotransformation is efficient, so effects of mutations engineered in vitro can readily be tested by reintroducing the altered genes into an appropriate mutant strain together with a selectable marker. Because nearly all nuclear transformation events result from apparently random integration of the introduced plasmid into chromosomal DNA, it is possible to generate large numbers of insertional mutants. The subsequent isolation of the disrupted genes by virtue of the molecular tag provides a very powerful means for cloning genes with known mutant phenotypes. Alternatively, since nuclear transformation is so efficient, genes with good selectable phenotypes can be isolated by complementing appropriate recessive mutations with pools of DNA from an indexed genomic library. Recently, expression of eubacterial genes that confer antibiotic resistance to C. reinhardtii has been reported; these genes should be useful as dominant selectable markers in any genetic background. However, transformants are recovered with these bacterial genes at only 1 % the rate obtained by transformation with C. reinhardtii genes, and in about half the cases the introduced genes are silenced under nonselective conditions, sometimes a problem with C. reinhardtii genes as well. Although an active homologous recombination system allows efficient recombination between simultaneously introduced plasmids, the rate of gene-targeted homologous integration events is very low. Further understanding of the factors that limit the expression of reintroduced genes and the rate of gene-targeting could lead to substantial improvements in the capability of manipulating the nuclear genome and of generating or phenocopying mutations corresponding to cloned genes.