shRNA gene silencing Human - Islets of langerhans Negative control (scrambled) lentiviral particles
Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure,
and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called
RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors.
Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging
shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on
which splice variants of the gene are desired. One should use the latest algorithms and choose
at least two different sequences, targeting different regions, in order to have confidence in
overcoming off-target effects. A BLAST search after selecting potential design will eliminate
potential off-target sequences. For the second challenge, sequencing the vector using primers
for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on
agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is
important to check the viral titer before transduction. Finally, using a marker in the lentiviral
vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression
can help in determining efficacy of transduction and shRNA on its target site.