Biology Reference
In-Depth Information
A
Ectopic expression of miRNAs
i. EP elements :
P
element with UAS
sites for Gal4-mediated activation
Gal4
UAS
Misexpression of endogenous miRNA locus
Different Gal4 lines provides tissue specificity
ii. UAS-miRNA transgenes
Gal4
Terminator
UAS
Engineered construct
Can include marker transgene (e.g., DsRed)
B
Expression of miRNA sponges
Gal4
UAS
miRNA binding sites
Terminator
Sequester miRNA from endogenous targets
Creates partial loss-of-function
Can include marker transgene (e.g., GFP)
Figure 8.2
Modulating miRNA activity using the Gal4/UAS system. Gal4/UAS is a
modular system for overexpressing transcripts under the control of the yeast Gal4
transcription factor, via “upstream activating sequences” (UASs) linked to a target
gene. Many spatially/temporally restricted Gal4 transgenes are available. (A) Ectopic
expression of miRNAs. As with protein-coding genes, ectopic expression of miRNAs
can lead to dominant phenotypes. Many miRNA loci were discovered by genetic
screens of collections of EP elements (i) transposons bearing UAS sites, which permit
activation of neighboring genes in a Gal4-dependent manner. Engineered UAS-
miRNA transgenes (ii) often contain the hairpin flanked by
200nt of endogenous
sequence, as well as a marker transgene that reports on miRNA-expressing cells.
(B) Inducible miRNA sponge transgenes, bearing multiple copies of a given miRNA
target site, enable tissue-specific knockdown of miRNA activity.
to generate specific miRNA mutants. Nearly all miRNA alleles are straight
deletions, but having a range of point mutations could provide interesting
insights, as with protein-coding genes.
In the case of
mir-278
,
its
