Biomedical Engineering Reference
In-Depth Information
(HCV), infl uenza virus, West Nile virus, and human papillomavirus
(HPV) [
1
-
5
]. These studies have provided important insights into
not only individual cell proteins but also pathways, compartments,
and large protein networks that facilitate virus uptake. Genome-
wide RNA interference screens will undoubtedly remain an impor-
tant tool for virologists to study both existing and newly emerging
pathogens for decades to come.
The increasing use of high-throughput genomics had also high-
lighted the need for careful and thorough hit validation during the
follow-up stages of the screening process. Illustrating the impor-
tance of hit validation are the large and well-documented diver-
gences in lists of required genes reported by investigators studying
the same pathogen [
6
]. Although some of the discrepancies can be
attributed to variations in assay design or inherent diffi culties in
interpreting the effects of siRNAs, the surprising lack of agreement
among data from different laboratories further emphasizes the
necessity for a multipronged experimental strategy to confi rm the
chief fi ndings from each screen [
7
]. Here we briefl y outline a num-
ber of experimental strategies to validate siRNA screen hits
(Subheading
1
) and describe in detail PLA- and fl ow cytometry-
based approaches to track virus entry and quantitate the extent of
virus disassembly (Subheadings
3.1
and
3.2
, respectively).
Although transfection of siRNAs into cells (or expression of
shRNAs) is an effi cient method to downregulate target gene
expression, some siRNAs bind to unintended mRNA transcripts
and infl uence their stability or translation [
8
]. Knockdown studies
that generate the same phenotype with more than one siRNA spe-
cifi c for non-overlapping sequences within the same target tran-
script can minimize the likelihood that a particular phenotype is
due to off-target effects. Because it is unlikely that multiple sequence-
divergent siRNAs will produce identical off-target effects, a pheno-
type (such as inhibition of virus infection) is likely to be valid when
it is reproduced by two or more different siRNAs (Fig.
1
).
In addition to testing a number of siRNAs to confi rm the role
of a gene in virus entry, validation of a phenotype can be achieved
by the expression of an siRNA- or shRNA-resistant cDNA of the
gene of interest (Fig.
2
). In this approach, silent mutations are
introduced into the cDNA of the targeted mRNA at the seed
sequence recognized by the siRNA. If the delivery of this construct
into cells eliminates the effect of the siRNA, this phenotype can be
ascribed with confi dence to target gene knockdown.
Pharmacological inhibition of protein function is a comple-
mentary approach to the use of genetic techniques to validate
siRNA hits. The advantages of pharmacological inhibitors include
the ease of use (no transfection or virus-mediated delivery is
required), targeted inhibition of protein activity, and the ability to
control the duration and dose of treatment. However, a major
disadvantage of chemical compounds is their well-documented
1.1 Screen Hit
Validation
