Synthetic Biology Parts, Devices and Applications

152 7 Splicing and Alternative Splicing Impact on Gene Design

either the 5′ splice site or the branch point. Maximal regulation (16-fold) was

observed with a construct in which the 5′ splice site was masked by intramolecu-

lar base pairing when placed within the closing stem of the aptamer. This blocked

the accessibility of the 5′ splice site to the U1 snRNP. The dynamic range of regu-

lation was increased by additionally inserting a second aptamer-containing intron.

Another programmable control device expands the possibility to engineer

alternative splicing by being triggered by the presence of specific protein binding

to an aptamer located in the intronic sequence. This approach has been success-

fully used to rewire both the Wnt and nuclear factor κB signaling pathways in

mammalian cells [254]. In plants a naturally occurring rRNA-mimicking struc-

ture was used to regulate cassette exon splicing in response to the expression of

a ribosomal protein. By using an engineered variant of the RNA structure from

another plant species highly efficient, orthogonal gene activation could be

achieved in Nicotiana benthamiana [255].

w/o intr

on

w/o intr

on

bgl intr

on 2

bgl intr

on 1

Synthetic intr

on

Endogenousintron

MAX

(a)

(b)

3

2

1

0

Re

lativ

e lucif

erase e

xpr

ession

n.d.

β-Actin

Figure 7.4 Influence of introns on gene expression. (a) Western blot analyses after

overexpression of MAX protein either lacking (w/o) or containing one endogenous intron in

the ORF. Overexpression was performed in HeLa cells and protein was isolated after 24 h.

β-Actin was used as a loading control. (b) Firefly luciferase reporter gene constructs without

(w/o) intron or containing the β-globin introns 1, 2 or a synthetic intron. Firefly luciferase

activity was measured in triplicates 24 h after transfection of HeLa cells using enilla luciferase

as transfection control. bgl = β-globin, n.d. = not determined.