title: A robust protocol for efficient generation, and genomic characterization of insertional mutants ofChlamydomonas reinhardtii.
abstract: BACKGROUND
  Random insertional mutagenesis ofChlamydomonas reinhardtiiusing 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA.
authors.forename: Steve V
authors.initials: SV
authors.lastname: Pollock
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA.
authors.forename: Bratati
authors.initials: B
authors.lastname: Mukherjee
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA.
authors.forename: Joanna
authors.initials: J
authors.lastname: Bajsa-Hirschel
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA.
authors.forename: Marylou C
authors.initials: MC
authors.lastname: Machingura
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA.
authors.forename: Ananya
authors.initials: A
authors.lastname: Mukherjee
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305 USA.
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Grossman
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA.
authors.forename: James V
authors.identifier: 0000-0002-3652-5293
authors.initials: JV
authors.lastname: Moroney