7.8 Splicing and Synthetic Biology 151stability, and the translatability of the mRNA (reviewed in [239]). As a conse-
quence, the expression of a gene can change dramatically depending on the
presence or absence of introns.
Gene expression is usually reduced upon removal of endogenous introns [240].
So the maintenance of introns can lead to considerable higher protein expression,
for example, for overexpression studies [241]. It is exemplified in Figure 7.4a,
where the expression of the MAX (Myc associated factor X) protein from an
intronless cDNA was compared with a cDNA retaining one endogenous intron.
Also the heterologous expression of transgenes can be increased significantly
by adding just a single generic intron [242–245]. The extent of the effect depends
on intron identity, intron position within the gene, and the surrounding exonic
sequences. Placing the same intron between different exons may yield opposing
results [241, 246]. The insertion of intron 2 of the β-globin gene into a firefly
luciferase reporter gene increased its expression 3-fold, the insertion of a syn-
thetic intron only 1.5-fold (see Figure 7.4b). In contrast, insertion of β-globin
intron 1 led to undetectable reporter activity. Therefore, the inability of β-globin
intron 1 to confer efficient splicing in a heterologous context is apparently due to
its weak splicing signals and missing enhancing sequences in the artificial con-
text [247]. The insertion of two short introns from the immunoglobulin heavy
chain into both a green fluorescent protein (GFP) reporter gene and a Cre recom-
binase cDNA increased gene expression up to 30-fold in CHO cells. These
introns were chosen because they were short, compatible with high levels of gene
expression, and without evidence of containing regulatory sequences [248]. In
line with this approach, several commercially available expression vectors also
contain short synthetic introns in their 5′ UTRs known to enhance the stability
of the mRNA by influencing polyadenylation [249].
7.8.2 Control of Splicing by Engineered RNA-Based Devices
The controlled removal of intronic sequences offers the possibility to engineer
user-defined gene expression systems. RNA-based control devices generally
couple in vitro selected RNA aptamers as sensory domains to functional RNA
domains (like a rbs, splice site, or a ribozyme). By modulating the accessibility of
elements essential for splicing, such as the 5′ splice site, the branch point, or the
3 ′ splice site, engineered riboswitches have been shown to control both constitu-
tive and alternative splicing.
In a pioneering study, a theophylline-binding aptamer was inserted close to a
3 ′ splice site. The addition of the ligand theophylline resulted in a 4-fold reduc-
tion of gene expression in an in vitro splicing assay [250, 251]. The data indicated
that theophylline binding specifically blocked the recognition of the 3′ splice site.
This aptamer was also used to modulate splicing efficiency by including the
branch point sequence into the aptamer sequence [252]. In the presence of theo-
phylline, the downstream exon was skipped twice more often than in its absence,
indicating that engineered riboswitches can also modulate and therefore investi-
gate the impact of alternative splicing.
A tetracycline-binding aptamer was used to regulate pre-mRNA splicing in
yeast [253]. The aptamer was inserted into a yeast intron in close proximity to