3 Methods
3.1 Cloning
of Plasmid Constructs
The visualization of RNA with the MS2 system involves the
co-expression of MSL-tagged RNA together with fluorescent
protein-tagged MCP. Here we describe the insertion of 12 copies
of the MSL into the 3
0 UTR sequences encoded by the Gateway
®
(see Note 3)-compatible plant expression vector pMDC32 to generate a MSL-tagged destination vector, and its application as destination vector for the expression of TMV MP fused to mRFP (MP:
mRFP) and its MSL-tagged mRNA. The plasmid vector for the
expression of NLS-tagged MCP fused to GFP (pB7-NLS:MCP:
GFP) has been described elsewhere [35], and this plasmid is available upon request.
3.1.1 Cloning
of the Destination Vector
pMDC32-GW-12xMSL
1. Use a high-fidelity polymerase and the primers MS2-12SLfw
and MS2-12SLrev to amplify a 708 bp long DNA fragment
encoding 12 copies of the MSL of the plasmid pSL-MS2-12
(available at http://www.addgene.org/27119/). The primer
sequences contain PacI and SacI restriction sites (underlined)
required for further cloning steps.
2. Verify the expected size of the amplified DNA fragment by
agarose electrophoresis and purify the DNA fragment from
the gel using a DNA gel extraction kit following the manufacturer’s instructions.
3. Insert the purified DNA fragment into a PCR cloning vector
(e.g., pGem
® -T Easy, Promega) using T4 DNA ligase according to the manufacturer’s instructions. Transform competent
E. coli cells with 1.0–3.0 μL of the ligation mix and plate them
on petri dishes with antibiotic selection (100 μg/mL ampicillin
for pGem
® T-Easy). Before plating the cells, distribute 5 μL of
0.5 mM IPTG and 25 μL of 80 μg/mL X-Gal onto the plate.
Incubate the plates with bacteria overnight at 37
C.
4. Isolate bacteria from 4 to 5 white colonies and propagate them
overnight in 15 mL falcon tubes containing 4.0 mL of liquid
LB medium with antibiotics (100 μg/mL ampicillin for
pGem
® -T Easy) at 37
C with agitation (180–200 rpm).
5. Harvest bacteria by centrifugation and isolate plasmid DNA
using a plasmid purification kit and following the manufacturer’s instructions. Verify the presence and correct size of the
inserted DNA by PCR amplification (using universal primers
homologous to vector sequences, e.g., T7 and SP6 primers
homologous to T7 and SP6 promoter sequences present in
pGem
® -T Easy) and agarose gel electrophoresis. Confirm the
absence of mutations by DNA sequencing and select one of the
verified plasmid clones to proceed with the following steps.
RNA Imaging in Plants Using MS2
111
3.1 Cloning
of Plasmid Constructs
The visualization of RNA with the MS2 system involves the
co-expression of MSL-tagged RNA together with fluorescent
protein-tagged MCP. Here we describe the insertion of 12 copies
of the MSL into the 3
0 UTR sequences encoded by the Gateway
®
(see Note 3)-compatible plant expression vector pMDC32 to generate a MSL-tagged destination vector, and its application as destination vector for the expression of TMV MP fused to mRFP (MP:
mRFP) and its MSL-tagged mRNA. The plasmid vector for the
expression of NLS-tagged MCP fused to GFP (pB7-NLS:MCP:
GFP) has been described elsewhere [35], and this plasmid is available upon request.
3.1.1 Cloning
of the Destination Vector
pMDC32-GW-12xMSL
1. Use a high-fidelity polymerase and the primers MS2-12SLfw
and MS2-12SLrev to amplify a 708 bp long DNA fragment
encoding 12 copies of the MSL of the plasmid pSL-MS2-12
(available at http://www.addgene.org/27119/). The primer
sequences contain PacI and SacI restriction sites (underlined)
required for further cloning steps.
2. Verify the expected size of the amplified DNA fragment by
agarose electrophoresis and purify the DNA fragment from
the gel using a DNA gel extraction kit following the manufacturer’s instructions.
3. Insert the purified DNA fragment into a PCR cloning vector
(e.g., pGem
® -T Easy, Promega) using T4 DNA ligase according to the manufacturer’s instructions. Transform competent
E. coli cells with 1.0–3.0 μL of the ligation mix and plate them
on petri dishes with antibiotic selection (100 μg/mL ampicillin
for pGem
® T-Easy). Before plating the cells, distribute 5 μL of
0.5 mM IPTG and 25 μL of 80 μg/mL X-Gal onto the plate.
Incubate the plates with bacteria overnight at 37
C.
4. Isolate bacteria from 4 to 5 white colonies and propagate them
overnight in 15 mL falcon tubes containing 4.0 mL of liquid
LB medium with antibiotics (100 μg/mL ampicillin for
pGem
® -T Easy) at 37
C with agitation (180–200 rpm).
5. Harvest bacteria by centrifugation and isolate plasmid DNA
using a plasmid purification kit and following the manufacturer’s instructions. Verify the presence and correct size of the
inserted DNA by PCR amplification (using universal primers
homologous to vector sequences, e.g., T7 and SP6 primers
homologous to T7 and SP6 promoter sequences present in
pGem
® -T Easy) and agarose gel electrophoresis. Confirm the
absence of mutations by DNA sequencing and select one of the
verified plasmid clones to proceed with the following steps.
RNA Imaging in Plants Using MS2
111