Advantages of the TargeTron System
Numerous characteristics of the group II retrohoming mechanism make it attractive for genetic manipulation. First, retrohoming is highly efficient, enabling creation of knockouts without the need for a selection marker. To complement the current kanamycin-based TargeTron system (TA0100), Sigma plans to release a series of TargeTron pACD4 vectors that do not insert antibiotic markers. These will allow researchers to expedite the creation of multiple knockouts.
Second, the introns have a minimal dependence on host factors, making them applicable to a broad range of bacteria. To date, the TargeTron system has been validated in a wide range of bacteria including: E. coli, Staphylococcus aureus, Clostridium, Shigella flexneri, and Salmonella typhimurium. The system may be modified for use in additional organisms.
In addition to site-specific knockouts, random chromosomal insertion libraries can be created. This approach allows essential and nonessential genes to be identified by PCR and sequencing. Once a desired random insertion is identified by PCR, the gene-specific intron sequence can be cloned and used to re-create the site-specific knockout in hosts with alternative genetic backgrounds.
This eliminates the need for maintaining large clonal libraries to isolate random insertion events and also eliminates the need for phage-based transfer of mutations between strains.
In addition to knocking genes out by insertional mutagenesis, the TargeTron system can be modified to deliver heterologous DNA. This feature allows the use of retrotransposition-activated selectable markers (RAM).
The currently available TargeTron system uses the same RAM approach, but with a kanamycin marker. This feature is not limited to the use of selectable markers and was recently used to examine the use of the TargeTron system to introduce therapeutic sequences site specifically into mutant genes.
The ability to target specific genomic regions and deliver heterologous DNA is a powerful combination allowing for the study of various chromosomal regions using promoters, reporters, and other genetic elements.
In summary, compared to existing knockout methodologies for prokaryotes, the TargetTron system allows efficient, targeted disruption of the gene of interest in a wide range of bacteria. It has significant applications in genetic engineering and metabolic engineering in systems biology and functional genomics.