5. Digest PCR fragment with restriction enzymes (primers are
designed to contain recognition sites for appropriate restriction
enzymes, which here can subsequently be used to clone the
PCR product). Gel purify digested PCR fragment after electrophoresis in a 1.2% TBE agarose gel.
6. Digest 1 μg of pENTR11 plasmid with appropriate restriction
enzymes. Gel purify the digested plasmid.
7. Set up a 10 μL ligation reaction by mixing 100 ng of digested
pENTR11 plasmid, 3Â molar excess of the digested PCR
product, 1 μL of 10Â T4 DNA ligase buffer, appropriate
amount of water, and 1 μL (5 U) of T4 DNA ligase. Incubate
the reaction at room temperature for 2–4 h.
8. Chemically competent TOP10 E. coli cells are transformed
with the ligation reaction. Spread bacteria onto an LB agar
plate containing 50 μg/mL of kanamycin. Incubate the plates
overnight at 37
C.
9. Screen the appearing colonies by colony PCR.
10. Purify plasmids from colonies carrying inserts of the expected
size and verify identity of the fragments by sequencing.
3.2.2 Transfer of miRNA
Genes into
a Gateway-Compatible
Binary Expression Vector
1. Set up LR recombination reaction by mixing 1 μL (50 ng) of
entry plasmid (pENTR11 containing the cloned miRNA gene)
with 1 μL (50 ng) of plant binary destination vector (e.g.,
pK7WG2D). Finally, add 0.5 μL of LR clonase. Incubate the
reaction at room temperature for 1–12 h.
2. Chemically competent TOP10 E. coli cells are transformed
with the recombination reaction. Spread bacteria onto an LB
agar plate containing 100 μg/mL of spectinomycin. Incubate
the plates overnight at 37
C.
3. Screen the appearing colonies by colony PCR.
4. Purify plasmids from colonies and verify correct structure of the
plasmid by restriction digestion.
3.3 Generation
of Sensor Constructs
A crucial component of the reporter system is the sensor plasmid.
AGOs can repress gene expression via miRNA-binding sites located
either in the 3
0 UTR or the ORF, with a considerably stronger effect
on the latter. Although the underlying mechanisms are still not
completely elucidated, this may be related to the divergent repression mechanisms acting on the topologically different target sites:
inhibition of translation initiation on 3
0 UTR sites versus steric
hindrance on ORF sites. In animals, miRNA-binding sites are
generally present in the 3
0 UTR, while in plants the ORF location
is predominant. Our comparative assessment revealed that miRNAbinding sites in the ORF behaved more like binary switches, while
in the 3
0 UTR they bestowed graded response to the sensor.
An In Vivo Reporter System for RNA Silencing
439
designed to contain recognition sites for appropriate restriction
enzymes, which here can subsequently be used to clone the
PCR product). Gel purify digested PCR fragment after electrophoresis in a 1.2% TBE agarose gel.
6. Digest 1 μg of pENTR11 plasmid with appropriate restriction
enzymes. Gel purify the digested plasmid.
7. Set up a 10 μL ligation reaction by mixing 100 ng of digested
pENTR11 plasmid, 3Â molar excess of the digested PCR
product, 1 μL of 10Â T4 DNA ligase buffer, appropriate
amount of water, and 1 μL (5 U) of T4 DNA ligase. Incubate
the reaction at room temperature for 2–4 h.
8. Chemically competent TOP10 E. coli cells are transformed
with the ligation reaction. Spread bacteria onto an LB agar
plate containing 50 μg/mL of kanamycin. Incubate the plates
overnight at 37
C.
9. Screen the appearing colonies by colony PCR.
10. Purify plasmids from colonies carrying inserts of the expected
size and verify identity of the fragments by sequencing.
3.2.2 Transfer of miRNA
Genes into
a Gateway-Compatible
Binary Expression Vector
1. Set up LR recombination reaction by mixing 1 μL (50 ng) of
entry plasmid (pENTR11 containing the cloned miRNA gene)
with 1 μL (50 ng) of plant binary destination vector (e.g.,
pK7WG2D). Finally, add 0.5 μL of LR clonase. Incubate the
reaction at room temperature for 1–12 h.
2. Chemically competent TOP10 E. coli cells are transformed
with the recombination reaction. Spread bacteria onto an LB
agar plate containing 100 μg/mL of spectinomycin. Incubate
the plates overnight at 37
C.
3. Screen the appearing colonies by colony PCR.
4. Purify plasmids from colonies and verify correct structure of the
plasmid by restriction digestion.
3.3 Generation
of Sensor Constructs
A crucial component of the reporter system is the sensor plasmid.
AGOs can repress gene expression via miRNA-binding sites located
either in the 3
0 UTR or the ORF, with a considerably stronger effect
on the latter. Although the underlying mechanisms are still not
completely elucidated, this may be related to the divergent repression mechanisms acting on the topologically different target sites:
inhibition of translation initiation on 3
0 UTR sites versus steric
hindrance on ORF sites. In animals, miRNA-binding sites are
generally present in the 3
0 UTR, while in plants the ORF location
is predominant. Our comparative assessment revealed that miRNAbinding sites in the ORF behaved more like binary switches, while
in the 3
0 UTR they bestowed graded response to the sensor.
An In Vivo Reporter System for RNA Silencing
439