Electroporation had been used to introduce plasrnid pCaMVCAT into a wall-less Chlamydomonas reinhardtii strain. Different types of mutants were generated. Transformants were selected by their ability to grow on chloramphenicol containing agar plate as the pCaMVCAT harbours the gene coding for chlorarnphenicol acetyltransferase (CAT) which can inactivate chloramphenicol (CAM). Among these CAM resistant mutants, Cd²⁺-sensitive and Cd²⁺-resistant mutants were selected out for further characterization. The fate of pCaMVCAT introduced into the algal cells was also analyzed. It is discovered that the free plasmid, pCahNCAT, can replicate within the algal cells although the stability of the free plasmid declines on continuous subculturing of the mutants in selective medium. In three out of fiv...[ Read more ]

Electroporation had been used to introduce plasrnid pCaMVCAT into a wall-less Chlamydomonas reinhardtii strain. Different types of mutants were generated. Transformants were selected by their ability to grow on chloramphenicol containing agar plate as the pCaMVCAT harbours the gene coding for chlorarnphenicol acetyltransferase (CAT) which can inactivate chloramphenicol (CAM). Among these CAM resistant mutants, Cd²⁺-sensitive and Cd²⁺-resistant mutants were selected out for further characterization. The fate of pCaMVCAT introduced into the algal cells was also analyzed. It is discovered that the free plasmid, pCahNCAT, can replicate within the algal cells although the stability of the free plasmid declines on continuous subculturing of the mutants in selective medium. In three out of five analyzed mutants, a small amount of pCaMVCAT could be detected 15 months after it was introduced into the algal cells. In all five mutants, plasmid pCaMVCAT was found to integrate stably into their algal nuclear genome. The number of integration sites varies from one to six. Our results demonstrate that the electroporation of pCaMVCAT can be used for insertion mutagenesis of Chlamydomonas reinhardtii.