Figure 1

Methods to generate multi-copy clones. Schematic representation of some of the more common methods used to create multi-copy clones. Multiple selection markers can be used when a gene is integrated into the genome through a vector with a single selection marker. This method is limited to the number of selection markers available (either antibiotic or through complementation to auxotrophic genes). Additionally, each vector must be transformed sequentially and the labor associated with selection increases with each additional gene. In vitro multimerization uses the pAO815 vector that isolates an expression cassette containing the promoter, gene of interest and transcription terminator region and ligating this in a head-to-tail orientation into a linearized vector. Copy number is determined prior to integration into the genome. Direct selection on high concentrations of antibiotic uses a single transformation with a vector containing either G418 or Zeocin™ and selection directly onto high concentrations of the antibiotic. This results in jackpot colonies (over 10 copies of the gene) in less than 1% of all clones. Posttransformational vector amplification (PTVA) uses a single vector for transformation (containing either the G418 or Zeocin™ resistance marker). Selection is originally on a low concentration of the corresponding antibiotic, but the cells are increasingly subjected to higher concentrations. Only colonies that have multiple copies of the resistance gene (and therefore multiple copies of the heterologous gene) will be able to survive on the highest concentrations. Jackpot colonies are reported in 6% of all clones tested. Integration into the rDNA locus with PTVA utilizes the repeat sequence of the rDNA (appearing 16 times in GS115), which can prevent tandem head-to-tail integration. Multi-copy clones are generated using PTVA.