The unicellular microalga
Chlamydomonas reinhardtii (
C. reinhardtii) has been used as a reference model for numerous fields of research. Principle research areas are eukaryotic flagellar structure and function, basal bodies (centrioles), cell-cell recognition, cell cycle control, chloroplast biogenesis, phototaxis, nonphotochemical quenching, and especially photosynthesis for
C. reinhardtii can grow in the dark on an organic carbon (
e.g. acetate), and thus provides advantages over land plants (Harris, 2001; Peers
et al., 2009).
C. reinhardtii has a short life cycle, a sequenced genome (Merchant
et al., 2007), and a growing molecular toolbox for forward and reverse genetic studies, including transformation protocols, gene silencing (Kim and Cerutti, 2009; Molnar
et al., 2009), and fluorescent protein-tag (Rasala
et al., 2013). There are two commonly used methods for
C. reinhardtii transformation – electroporation and glass bead agitation. Electroporation is normally restricted to strains with cell wall, as it kills cell-wall-deficient strains effectively if without careful handling of osmosis. Electroporation also requires special instruments such as electroporator and cuvettes. In contrast, glass bead agitation uses simple lab equipment. The mild shear created by agitation in the presence of glass bead allows cell-wall-deficient strains to take up DNA. If glass bead method is to be applied to cell-wall strains, cells need to be treated with autolysin (
http://www.chlamy.org/methods/autolysin.html) to partially lyse the wall components. A pitfall of both methods is that the DNAs are often shortened by nuclease once entering the cells, making the downstream PCR-based genotyping of insertion site rather difficult. Here I describe an improved design of insertional mutagenesis used in (Tsai
et al., 2014), and the transformation protocol using glass bead as previously described in (Kindle, 1990) with minor modification. The putative mutants can be selected by autotrophic or antibiotic resistance markers, and the disrupted loci can be mapped by methods such as plasmid rescue (Peers
et al., 2009) and SiteFinding PCR (Tan
et al., 2005).