Uptake and integration of foreign DNA in Chlamydomonas monoica promoted by glass bead vortexing
Cesar Fuentes and Karen VanWinkle-Swift
Dept. Biol. Sciences, Northern Arizona University, Flagstaff AZ 86011-5640, USA
Gene silencing is often a major obstacle in developing transformation protocols, especially those based on the expression of foreign genes. Expression of foreign genes differs among species with some exhibiting strong expression while others show little or no expression despite the presence of the foreign gene. In order to avoid gene silencing issues in Chlamydomonas monoica, we have used an approach focusing on insertional mutagenesis following glass-bead-induced uptake of exogenous foreign DNA. A previously isolated cell wall-less strain (cw-1) from C. monoica was used as the recipient. The transforming DNA included plasmid vectors (pSL17, pSI103, p681 and p699) containing the eubacterial aphVIII (paromomycin resistance) or aadA (spectinomycin resistance) genes driven by C. reinhardtii controlling elements. Chlorate resistant insertional mutants were selected and assayed for the presence of the foreign gene by PCR and Southern Blot.Unstable foreign gene expression, as described for the aadA gene in C. reinhardtii, was verified by plating the putative transformants at high cell density on antibiotic medium. The preferred conditions for transformation were established by varying the concentration of the transforming DNA and polyethylene glycol (PEG) as well as other conditions that have been demonstrated to enhance transformation efficiency.These include glass bead size, time of agitation, foreign DNA confirmation (linearized vs. non-linearized), recovery time, and the absence/presence of carrier DNA. The glass bead vortexing technique provides more reproducible and efficient transformation than we previously reported for electroporation. (Supported by NIH grant 2 R15 GM071374-02).
e-mail address of presenting author: cf5@nau.edu