Manipulating Levels of Specific MicroRNAs in Mammalian Cells - MicroRNAs as Tools in Biopharmaceutical Production - page 115

8.3 Stable Expression of miRNAs or Inhibitors

While synthetic miRNA mimics and inhibitors may be useful to verify or identify
candidate miRNAs for up or down regulation in transient assays, stable expression is
preferred for recombinant protein production. Even the most stable modified oligonu-
cleotide will be diluted to an ineffective level as cells divide. Furthermore, it is neither
practical nor regulatory compliant in production to re-transfect cells every few days
with synthetic miRNAs or inhibitors. For stable expression, constructs encoding
miRNAs or miRNA inhibitors may be inserted into and expressed from the genome.
Both plasmid and viral vectors are used to deliver miRNA and antimiR expression
constructs. To express miRNAs, the DNA sequence encoding the miRNA hairpin
precursor along with 100-200 bases upstream and downstream have been cloned be-
tween an appropriate RNA polymerase II (Pol II) promoter and a 3 -polyadenylation
sequence. However, since miRNAs can be regulated post-transcriptionally (Lee et al.
2008 ; Thomson et al. 2006 ), processing of a miRNA into its mature, active form may
be inhibited depending on cell line and growth conditions. As an alternative to the
natural precursor, artificial miRNAs are often used. To construct an artificial miRNA,
the primary sequence of an expressed miRNA is used as a scaffold or backbone, with
the stem sequence replaced by that for the miRNA of interest (Amendola et al. 2009 ).
Such artificial miRNAs are processed like the parental miRNA used for the scaffold
(Zeng et al. 2002 ). miRNA hairpin sequences have also been inserted into an artifi-
cial intron in green fluorescent protein (GFP) for expression (Amendola et al. 2009 ;
Qiu et al. 2008 ). GFP can be used to report successful transfection, transcription,
and processing, and fluorescing cells can be isolated by Fluorescence Activated Cell
Sorting (FACS).
Most miRNA expression constructs are transcribed from Pol II promoters, analo-
gous to their natural counterparts. Viral promoters such as cytomegalovirus (CMV)
are used for high level expression, while the phosphoglycerate kinase (PGK) pro-
moter is often used for more moderate expression or in cells that suppress or silence
viral promoters. Even higher levels of miRNA may be expressed from an RNA poly-
merase III (Pol III) promoter such as U6 (Zhou et al. 2008 ). Use of an inducible
promoter, so that expression of the miRNA can be turned on or off at an appropriate
stage, such as when the cells reach a desired density for recombinant protein produc-
tion, may also be beneficial. In fact, conditional expression may be essential if the
miRNA's activity or its loss is detrimental to cell viability or growth. Tet-inducible
promoters, which can be turned on or off by adding or removing doxycycline from
the culture medium, are commonly used to restrict expression to a desired growth
phase or time-frame (Gossen and Bujard 1992 ).
Pre-made lentiviral constructs for expressing human and mouse miRNAs are
available from several commercial sources. In addition, vectors with many of the
features mentioned above are commercially available for clone-your-own miRNA
expression, and several companies offer custom-made miRNA expression constructs
as a service.
Stable inhibition of miRNA activity can be achieved by expressing transcripts that
contain miRNA binding sites, i.e., sequences complementary to the miRNA, to act as