Open AccessDNA-mediated transformation of Chlamydomonas reinhardi cells: use of aminoglycoside 3'-phosphotransferase as a selectable marker.
Author(s) -
Seyed E. Hasnain,
Elias K. Manavathu,
WaiChoi Leung
Publication year - 1985
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.5.12.3647
Subject(s) - biology , selectable marker , microbiology and biotechnology , plasmid , southern blot , kanamycin , replicon , chlamydomonas , transformation (genetics) , genetics , gene , mutant
Using a modified vector, we developed a method for DNA-mediated transformation of Chlamydomonas reinhardi with increased efficiency. The vector contained the yeast 2 microns origin of replication as a heterologous replicon. The aminoglycoside 3'-phosphotransferase (APH) gene linked to the simian virus 40 early promoter was used as an antibiotic selectable marker. The C. reinhardi transformants were resistant to 12 micrograms of G418 or 150 micrograms of kanamycin per ml. A quick-blot mRNA analysis demonstrated the presence of RNase-sensitive transcripts from the APH gene in the transformants, suggesting that the acquisition of antibiotic resistance was due to the expression of the APH gene. Southern blot analysis revealed the presence of free plasmid DNA in the transformant. The transforming vector was recovered by transforming recipient bacteria with the total DNA extracted from the C. reinhardi transformant.