Selectable marker recycling in the chloroplast

Nicolas Fischer, Otello Stampacchia, Kevin Redding, Jean David Rochaix

Research output: Contribution to journalArticle

128 Citations (Scopus)

Abstract

The bacterial gene aadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of the aadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking the aadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which the aadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas the aadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse the aadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.

Original languageEnglish
Pages (from-to)373-380
Number of pages8
JournalMolecular and General Genetics
Volume251
Issue number3
DOIs
Publication statusPublished - Jan 1 1996

Keywords

  • Chlamydomonas reinhardtii
  • Chloroplast transformation
  • Recombination
  • aadA

ASJC Scopus subject areas

  • Genetics

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