A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

• Published in: Plant methods 2017-04, Vol.13 (1), p.22-22, Article 22
• Main Authors: Pollock, Steve V, Mukherjee, Bratati, Bajsa-Hirschel, Joanna, Machingura, Marylou C, Mukherjee, Ananya, Grossman, Arthur R, Moroney, James V
• Format: Article
• Language: English
• Subjects: Acclimation; Acclimatization; Adapters; Air conditioning; Algae; Amplification; Antibiotic resistance; Antibiotics; Asymmetry; Atmosphere; Bacteria; Biochemistry; Biological activity; Carbon concentrating mechanism; Carbon dioxide; Chlamydomonas Genome; Chlamydomonas reinhardtii; Chlorophyll; Coding; Cyc6 gene; Deoxyribonucleic acid; Dissection; DNA; DNA polymerase; Drug resistance; Electroporation Cuvette; Enzymes; Eukaryotes; Experimental design; Fragmentation; Fungi; Gene expression; Gene mapping; Genetic screening; Genetic transformation; Genetics; genome; Genomes; Genomics; Homology; Insertional Mutagenesis; Insertional Mutant; Inserts; Kinases; Methodology; Methods; Mutagenesis; Mutants; Nitrogen; Nuclei; Nucleotide sequence; Optimization; Paromomycin; Photosynthesis; Plants; Polymerase chain reaction; Position (location); Prokaryotes; Proofreading; Ribonucleic acid; RNA; Studies; Success; Sulfur; Temperature effects; Transcription
• ISSN: 1746-4811; 1746-4811
• DOI: 10.1186/s13007-017-0170-x

Random insertional mutagenesis of using drug resistance cassettes has contributed to the generation of tens of thousands of transformants in dozens of labs around the world. In many instances these insertional mutants have helped elucidate the genetic basis of various physiological processes in this model organism. Unfortunately, the insertion sites of many interesting mutants are never defined due to experimental difficulties in establishing the location of the inserted cassette in the Chlamydomonas genome. It is fairly common that several months, or even years of work are conducted with no result. Here we describe a robust method to identify the location of the inserted DNA cassette in the Chlamydomonas genome. Insertional mutants were generated using a DNA cassette that confers paromomycin resistance. This protocol identified the cassette insertion site for greater than 80% of the transformants. In the majority of cases the insertion event was found to be simple, without large deletions of flanking genomic DNA. Multiple insertions were observed in less than 10% of recovered transformants. The method is quick, relatively inexpensive and does not require any special equipment beyond an electroporator. The protocol was tailored to ensure that the sequence of the Chlamydomonas genomic DNA flanking the random insertion is consistently obtained in a high proportion of transformants. A detailed protocol is presented to aid in the experimental design and implementation of mutant screens in Chlamydomonas.