AROs targeted to TBK1 knock down RNA expression across multiple
locations on exon 14.
To evaluate whether AROs could knock down the expression of other genes,
we designed AROs against the TBK1 gene (Figure 3A). We targeted exon 14
because it contained multiple ASF/SF2 sites according to ESE finder
(Cartegni et al., 2003) and because its moderate size (122 bp) allowed
us to evaluate multiple AROs targeting the same exon, as diagrammed in
Figure 3A. To investigate whether AROs act in a location-specific
manner, we designed 5 different AROs targeting exon 14 of TBK1 in 20
base pair segments and tested them in HaCat cells. All 5 AROs
significantly knocked down TBK1 expression (P =< 0.01) when
compared to the mock transfection carrier control. The largest knockdown
was seen in ARL-TBK1-O3-ARL (51%) and the smallest in TBK1-O5-ARL
(37%). These results indicate that all AROs targeting exon 14 of TKB1
were effective at gene knockdown.
As a negative control, we included the ALR-Oligo-ARL construct targeting
the KRT14 gene from the previous experiment. As expected, the
KRT14-targeting ARO did not decrease TBK1 transcript levels. Instead, we
observed a significant (P <= 0.01) increase in TBK1 gene
expression when KRT14 is knocked down. We hypothesize that the increase
in TBK1 expression is a downstream result of perturbation of KRT14
expression levels.
Our experiments do not seem to indicate any preference for targeting
oligo location along the length of the exon as all of the
oligonucleotides that targeted exon 14 of TBK1 showed similar knockdown
effects.