Regardless, one has to keep in mind that testing of both target site
constellations may become necessary to find the arrangement
which fits best to one’s purpose.
We have designed a GFP-Renilla luciferase fusion gene platform to test the effectiveness of miRNA-binding sites in the two
different constellations [6]. The use of the fusion protein provides
several advantages: (1) GFP serves as a tag to detect and quantify
the expression levels of the fusion protein by sensitive GFP antibodies in Western blotting; (2) it allows parallel visual monitoring
and quantitative assessment of silencing (by observing GFP fluorescence and measuring enzymatic activity of the Renilla luciferase)
(see Note 10); (3) to generate ORF sensors, the linker region
connecting the two functional domains of the fusion protein can
be used as a suitable position to insert miRNA-target sites without
disrupting the activities of either GFP or Renilla luciferase.
To properly assess gene silencing, the activity measured on a
wild-type sensor construct has to be compared to the activity
observed on a corresponding mutant sensor (Fig. 1a). The two
sensors differ from each other only by three point mutations. These
mutations are introduced into the miRNA-binding site at positions
10, 11 (flanking the AGO cleavage site), and 16 (the 3
0 supplementary region).
3.3.1 Generation
of miRNA Target Site Entry
Vectors
1. Generate the pENTR11-Renilla luciferase vector by inserting
the 982 bp long NcoI-NotI fragment of psiCHECK—bearing
the Renilla luciferase ORF—into pENTR11 by conventional
asymmetric sticky-end ligation.
2. Anneal tandem wild-type or mutant miRNA-binding site containing complementary oligonucleotides to produce doublestranded DNA with the miRNA-binding sites as follows:
(1) mix 1 nmole of each of the complementary oligonucleotides in 100 μL of 1Â Tango buffer; (2) heat the mixture to
100
C for 5 min, then turn off the heating block, and allow it
to cool down slowly to room temperature (usually overnight).
3. Digest 1 μg of the pENTR11-Renilla luciferase plasmid (see
above) with either XhoI-XbaI (to yield 3
0 UTR entry vector) or
BstBI (to yield ORF entry vector) (Fig. 2). Gel purify the
digested plasmid.
4. Mix 100 ng of digested pENTR11-Renilla luciferase plasmid
with 0.1 pmole of the annealed miRNA-binding site-containing oligonucleotides. Set up a 10 μL ligation reaction by adding
1 μL of 10 Â T4 DNA ligase buffer, appropriate amount of
water, and 1 μL (5 U) of T4 DNA ligase to the above mixture.
Incubate the reaction at room temperature for 2–4 h.
5. Chemically competent TOP10 E. coli is transformed by the
ligation reaction as above.
440
Ka ´ roly Fa ´ tyol
constellations may become necessary to find the arrangement
which fits best to one’s purpose.
We have designed a GFP-Renilla luciferase fusion gene platform to test the effectiveness of miRNA-binding sites in the two
different constellations [6]. The use of the fusion protein provides
several advantages: (1) GFP serves as a tag to detect and quantify
the expression levels of the fusion protein by sensitive GFP antibodies in Western blotting; (2) it allows parallel visual monitoring
and quantitative assessment of silencing (by observing GFP fluorescence and measuring enzymatic activity of the Renilla luciferase)
(see Note 10); (3) to generate ORF sensors, the linker region
connecting the two functional domains of the fusion protein can
be used as a suitable position to insert miRNA-target sites without
disrupting the activities of either GFP or Renilla luciferase.
To properly assess gene silencing, the activity measured on a
wild-type sensor construct has to be compared to the activity
observed on a corresponding mutant sensor (Fig. 1a). The two
sensors differ from each other only by three point mutations. These
mutations are introduced into the miRNA-binding site at positions
10, 11 (flanking the AGO cleavage site), and 16 (the 3
0 supplementary region).
3.3.1 Generation
of miRNA Target Site Entry
Vectors
1. Generate the pENTR11-Renilla luciferase vector by inserting
the 982 bp long NcoI-NotI fragment of psiCHECK—bearing
the Renilla luciferase ORF—into pENTR11 by conventional
asymmetric sticky-end ligation.
2. Anneal tandem wild-type or mutant miRNA-binding site containing complementary oligonucleotides to produce doublestranded DNA with the miRNA-binding sites as follows:
(1) mix 1 nmole of each of the complementary oligonucleotides in 100 μL of 1Â Tango buffer; (2) heat the mixture to
100
C for 5 min, then turn off the heating block, and allow it
to cool down slowly to room temperature (usually overnight).
3. Digest 1 μg of the pENTR11-Renilla luciferase plasmid (see
above) with either XhoI-XbaI (to yield 3
0 UTR entry vector) or
BstBI (to yield ORF entry vector) (Fig. 2). Gel purify the
digested plasmid.
4. Mix 100 ng of digested pENTR11-Renilla luciferase plasmid
with 0.1 pmole of the annealed miRNA-binding site-containing oligonucleotides. Set up a 10 μL ligation reaction by adding
1 μL of 10 Â T4 DNA ligase buffer, appropriate amount of
water, and 1 μL (5 U) of T4 DNA ligase to the above mixture.
Incubate the reaction at room temperature for 2–4 h.
5. Chemically competent TOP10 E. coli is transformed by the
ligation reaction as above.
440
Ka ´ roly Fa ´ tyol