Biology Reference
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from the start of the mature miRNA) from genomic DNA and introduce appropriate
restriction enzyme sites at both ends (restriction enzyme sites are introduced via the
primers generated for amplification). Generate compatible ends on the purified
PCR product and a reporter gene vector by restriction enzyme digestion. Ligate the
promoter fragment and the linearized vector and amplify the ligated product in
E. coli. For promoter-gfp fusion, two vectors, pPD95.70 and pPD95.75 from the
Fire Vector Kit, have been broadly used in the C. elegans community. Both of them
carry a S65C variant of the gfp gene and pPD95.70 has a SV40 nuclear localization
sequence (NLS) for nuclear localization of GFP. The NLS may help with observa-
tions of weak GFP signals but should not be used to represent the subcellular
localization of the miRNA. The gfp gene is linked to a 3 0 UTR derived from the
muscle myosin heavy chain gene unc-54, permissive for expression in all cell types.
Although conventional cloning is relatively time-consuming and depends on
restriction enzyme sites, it has the irreplaceable advantage of generation of a
reusable reporter gene construct. This construct can be verified by sequencing,
repeatedly amplified, stored for a long time, and easily subjected to further
subcloning.
2. PCR Fusion
To prepare the promoter-reporter fusion construct more quickly than conventional
cloning and escape the limitation of restriction enzyme sites, a two-step approach of
PCR can be used to fuse a miRNA promoter and a reporter gene, generating a linear-
formed DNA ready for transformation. Below, we describe the procedures for fusing
a miRNA promoter to the
3 0 UTR, derived from
gfp
reporter with an
unc-54
pPD95.75, by PCR fusion ( Fig. 2 B).
Prepare the following primers:
Primer A: promoter-specific outside forward primer
Primer A*: promoter-specific nested forward primer
Primer B: promoter-specific reverse primer with a 24-bp sequence complementary
to the MCS of the expression vector. (5 0 -AGTCGACCTGCAGGCATGCAAGCT-
promoter specific sequence-3 0 )
Primer C: gfp forward primer (5 0 -AGCTTGCATGCCTGCAGGTCG-3 0 )
Primer D: gfp reverse primer (5 0 -AAGGGCCCGTACGGCCGACTA-3 0 )
Primer D*: gfp nested reverse primer (5 0 -GGAAACAGTTATGTTTGGTATA-3 0 )
Perform PCR using primers A and B to amplify a DNA fragment from genomic
DNA that contains the promoter region and a 24-bp fragment overlapping with the
MCS of pPD95.75. Perform PCR using primer C and D to amplify the gfp::unc-54
fragment from pPD95.75, which includes the MCS and an artificial intron between
MCS and the gfp gene. Inclusion of the artificial intron will enhance reporter gene
expression. Purify the two PCR products by electrophoresis on an agarose gel and
recover the DNA from the corresponding bands. Mix 1-50 ng of each purified DNA
fragment and use nested primers A* and D* for PCR fusion. Purify the fused
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