Biomedical Engineering Reference
In-Depth Information
Since early applications of this approach, initially restricted to a few pooled
deletant strains, bar-code identification was carried out by their hybridization to
specific microarrays [38,61]. Nowadays, the use of NGS techniques is applicable as
a more quantitatively and qualitatively reliable identification of bar codes [62].
14.3.1 Competitive Growth Assay Based on Heterozygote Strains
HIP was one of the first assays taking advantage of the competitive growth strategy.
Thanks to this approach, using a unique treatment it is possible to assay the response of
6343 deletant strains, corresponding to a strain for every single yeast identified ORF.
Indeed, the heterozygote background makes the generation of yeast strains deleted
for essential genes possible and still viable thanks to the presence of at least one
copy of the gene. Bar codes that decrease in abundance after treatment with respect
to the control allow for identification of the strains deleted for genes whose product
is targeted by the condition tested. The feasibility and strength of the competitive
assay has been demonstrated by screening novel compounds [61,63]; this approach
has then been applied to a dissection of the mode of action of novel synthetic and
natural compounds [64-66].
When treating the heterozygote bar-coded collection with a molecule whose
molecular target is under investigation, it is necessary to use a sublethal concen-
tration of the compound studied. Indeed, such a concentration will maintain constant
the growth rate of strains bearing both copies of the target gene and at the same time
will lower the growth rate of the strain heterozygote for that gene. Nevertheless, such
worldly wisdom is sometimes not sufficient, and the gene dosage targeted should be
decreased further. Yan et al. proposed a solution for that problem by generating a bar-
coded haploid DAmP (decreased abundance by
m
RNA perturbation) collection [67].
This new approach is not based on gene deletion but, rather, makes use of the replace-
ment of the downstreamnoncoding sequence of the gene (3
-UTR) with nonfunctional
heterologous sequences. The replacement of such a region eliminates the sequences
that stabilize the translated
m
RNA—such as polyadenylation signals—thus causing
a reduced
m
RNA accumulation [68]. Only about 10% on average of the wild-type
protein levels is expressed, whereas expression of the gene selected in heterozygote
bar-coded strains is decreased to 50% with DAmP. By using different heterologous
nonfunctional sequences for replacement of different genes' 3
-UTR regions, every
gene targeted is uniquely identifiable, thus allowing for pooled treatment of the entire
collection.
14.3.2 Competitive Growth Assay Based on Haploid or Homozygous Strains
The competitive growth assay based on haploid or homozygous strains is analogous
to the assay based on heterozygote strains, with the exception that the strains are
completely deleted for nonessential genes in either haploid or diploid strains. The
idea driving this approach is the same as that of the HOP assay, but extended to
a genomic scenario. In the heterozygote strain-based competitive growth assay, the
genes targeted by the molecule are identifiable by the bar codes overrepresented after
