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As shown in
Fig. 2
C, amplify attB4-miRNA promoter (Pmir)-attB1.1R and
attB1.1-gfp::unc-54 3
0
UTR-attB2.1 fragments by PCR from source DNA (genomic
DNA and pPD95.75). Purify the PCR products by agarose gel electrophoresis.
Recombine attB4-Pmir-attB1.1R with pDONR-P4-P1R (Invitrogen) and attB1.1-
gfp::unc-54 3
0
UTR-attB2.1 with pDONR201 or pDONR221 (Invitrogen). This will
generate two entry vectors, pENTRY-Pmir and pENTRY-gfp. Recombine pENTRY-
Pmir and pENTRY-gfp with pDEST R4-R2 or pDEST-DD03 (
Dupuy et al., 2004
)
(available from Marc Vidal, Harvard University). pDEST-DD03 contains attR4 and
attR2 sites along with an unc-119 gene which is used in ballistic transformation
protocols (
Praitis et al., 2001
). All recombination reactions are according to man-
ufacturer
'
s instruction.
Q. Reporter-miRNA Target 3
0
UTR Fusion
In addition to determining the expression pattern of miRNAs during development,
reporter fusion constructs can be used to study the miRNA-mediated regulation of
target genes. Usually, target genes of miRNAs are identified by genetic screening or
predicted by computational analysis, which is mostly based on the assumption that
miRNAs associate with regulatory elements in the 3
0
UTR of target genes. An exper-
imental approach to verify a newly found or predicted miRNA/target pair is to
examine the expression of a reporter that is fused with the 3
0
UTR of a target gene.
For example, our group has used a lacZ reporter construct, Pcol-10::lacZ::lin-41
3
0
UTR, to observe the regulation of heterochronic gene lin-41 by let-7
(
Reinhart et al., 2000
). The actual let-7 binding sites on lin-41 3
0
UTR were also
verified by a series of mutagenesis experiments on such a reporter (
Ve l l a et al., 2004
).
Moreover, this Pcol-10::lacZ::lin-41 3
0
UTR reporter construct can be used as a tool to
evaluate the efficacy of let-7 in wild-type or let-7 mutant animals (
Chan and Slack,
2009; Reinhart et al.,2000
). In these studies, we use the promoter of collagen gene
col-10 to specifically express lacZ in hypodermal seam cells where let-7 is expressed.
A reporter-3
0
UTR fusion can be constructed by cloning strategies similar to that
for building a promoter-reporter fusion (see above and
Fig. 2
). A fixed and cell-
specific promoter can be chosen to drive the reporter expression for the purpose of
observation. Usually, a promoter of a housekeeping gene or a gene that is not linked
to miRNA regulation will be chosen, like the col-10 promoter. In other cases,
researchers will choose the promoter from the gene of interest along with its
3
0
UTR and examine the reporter expression in the specific spatiotemporal pattern
of the endogenous gene. The 3
0
UTR region of most genes is annotated in WormBase
(
Harris et al., 2010
) or can be inferred by putative polyadenylation signal (PAS) sites
(
Hajarnavis et al., 2004
) or by RNA-seq data available as part of the track for ALG-1
binding at the UCSC Genome Browser (
Hillier et al., 2009; Zisoulis et al., 2010
).
Putative miRNA binding sites can be predicted by algorithms derived from PicTar
(
Lall et al., 2006
), TargetScan (
Lewis et al., 2005
), or miRanda (
Enright et al., 2003
).
The presence of ALG-1 binding sites can also be used to prioritize 3
0
UTR sequences
to test for miRNA regulation by reporter analyses (
Zisoulis et al., 2010
).
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