Biomedical Engineering Reference
In-Depth Information
14.3 CHEMICAL GENOMICS AND
S. cerevisiae
All the aforementioned chemical genetics approaches are based on single-strain
assays. Indeed, in every procedure, identification of the genotype associated with a
selected phenotype is the fundamental request for the success of the investigation.
Actually, in their work setting the foundations for the HIP approach, Giaever et al.
also proposed a way to specifically recognize different deletant strains from a pool of
deletant strains [38]. The ability to identify the deletion of a specific strain is possible
because of the bar coding of the deletion. In more detail, every functional gene
is substituted for by the KanMX cassette, conferring resistance on the antifungal
kanamycin, flanked by two unique 20-bp sequences (bar codes) specific for the
deletion. The presence of these specific and identifiable tags makes possible the
culturing of pooled deletant strains, thus reducing the experimental variability of
the treatment while reducing the culturing efforts otherwise necessary for parallel
treatments. An additional advantage in using a competitive culture assay is the request
for less of the compound being studied (one to two orders of magnitude). A yeast bar-
coded knockout collection has been generated consisting of a complete set of deletion
strains, including haploid strains of both yeast mating types and heterozygous and
homozygous diploid deletions (Figure 14.8).
FIGURE 14.8
Available yeast bar-coded deletion collections. A functional gene is deleted
through substitution with a genetic cassette conferring the deleted strain kanamycin resistance
(
KanMX
). The presence of two deletion-specific 20-mer sequences flanking the
KanMX
cassette
allows for identification of the deletion. The deletion of a single gene copy in diploid cells
allows for the study of essential and nonessential genes; the deletion of both gene copies
in diploid cells, or of the unique copy in haploid cells, allows for the study of nonessential
genes only.
