Homologous recombination is the process by which similar DNA
sequences exchange information with one another. Since homologous recombination
is a rare event, various selection markers are used for isolation of
recombinants that have undergone homo-logous recombination. Though a variety of
positive selection markers exists, very few negative selection markers are
available, such as are necessary to select against random integrations and/or
eliminate marker genes. Currently, for example, the herpes simplex-thymidine
kinase (HSV-TK) gene is the primary negative selection marker used. This system,
however, uses a nucleoside analog for the selection, thereby making it
potentially able to produce undesirable mutations elsewhere in the genomic DNA.
Researchers at the Biodesign Institute of Arizona State
University have developed a novel negative selection marker for the rapid and
efficient isolation of genetic recombinants. This negative selection scheme
consists of using a fusion protein, GyrB-PKR, in conjunct-tion with the
antibiotic, coumermycin A1. Under selective conditions, this marker regulates
eukaryotic translation machinery leading to the shut off of protein synthesis.
This is further unique compared to other negative selection markers that act on
replicating DNA.
In this system, genetic recombinants are enriched in the
selective media through the loss of the negative selection marker. This system
therefore provides a superior method of isolating clones in a safe and effective
manner, without increasing the chances of generating alterations in the DNA
elsewhere in the genome.
Potential Applications
- Vaccine development
- Cellular system analysis
- Development of genetically modified
mammals
Benefits and Advantages
- No extraneous marker sequence is left in the genetic
locus of interest.
- Universal to all eukaryotic organisms
- Avoids the use of special cell lines or noxious mutagenic
chemicals that can be potentially damaging to genomic DNA
- Can be used with any system, both DNA and RNA, that
undergoes homologous recombination
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For more information about the inventor(s) and their
research, please see
Dr.
Jacob's directory webpage
