Biomedical Engineering Reference
In-Depth Information
competitive inhibitors. Two main types have been reported—“sponges” and “tough
decoys” (TuDs). Sponge constructs use multiple copies of a miRNA binding site to
“soak up” or sequester miRNAs so that they are not available to regulate their natural
targets (Care et al.
2007
; Ebert et al.
2007
; Gentner et al.
2009
). miRNA binding
sites are often placed in the 3
UTR of GFP or other reporter protein so that loss of
signal indicates sponge function. Sponges have been used to inhibit miRNA activity
in cultured cells, as well as in intact flies (Loya et al.
2009
). TuDs, on the other hand,
are a Pol III transcribed RNA with two opposing single-stranded miRNA binding
sites sandwiched between a double-stranded stem and a stem-loop. Although TuDs
have just two miRNA binding sites per molecule, they are transcribed by Pol III, the
enzyme which drives expression of some of the most abundant RNAs. In addition,
TuDs were designed for efficient nuclear export and their secondary structure was
optimized for inhibition (Haraguchi et al.
2009
). miRNA inhibition by TuDs has also
been demonstrated in various cell types (Lu et al.
2011
; Sakurai et al.
2011
). Sponge
and TuD structures with mismatches or additional bases inserted in the middle of the
miRNA binding sites inhibit miRNAs more effectively than perfect complements,
likely because these mismatched or bulged structures can not be cleaved by RISC.
Both published (Haraguchi et al.
2009
) and our own unpublished results suggest that
TuDs may inhibit miRNA activity more effectively than sponges, likely owing to a
higher expression by Pol III.
8.4
Targeted Gene Editing
In most published work to date, stably expressed miRNAs and miRNA inhibitors
were integrated semi-randomly into the genome. Semi-random integration by lentivi-
ral or plasmid delivered constructs can disrupt genes with desired functions or activate
those detrimental to recombinant protein expression. In addition, regions surround-
ing the insertion site influence transgene expression, such that expression can vary
or become silenced because of location. While integration at a specific site by ho-
mologous recombination (HR) is fairly efficient in mouse embryonic stem (ES) cells
(Capecchi
2005
), such targeted integration is inefficient in other species and cell
lines.
Engineered zinc finger nucleases (ZFNs) that target the desired integration site
can facilitate HR such that targeted integration is sufficiently efficient for cell line
engineering. ZFNs are chimeric proteins, with multiple zinc finger protein domains
fused to the non-specific nuclease domain of FokI restriction enzyme. The zinc finger
domains bind specific DNA sequences and FokI, which acts as a dimer, cuts DNA
between two ZFN pairs. In cells, DNA cleaved by the FokI nuclease is repaired by
non-homologous end-joining (NHEJ) or by HR. Providing a donor construct with
a gene of interest flanked by sequences homologous to those on either side of the
cut site can facilitate HR and lead to insertion of the donor construct (Fig
8.1
). This
type of targeted integration has been widely demonstrated for protein expression
(Carroll
2011
). Appropriate “safe-haven” sites for targeted integration, that is, sites
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