quant have a built-in plug-in converting CellProfiler outlines
for FISH-quant analysis. 3D gaussian fitting of the single spots
generates a text file containing the x,y position, brightness, and
number of spots identified in each cell. In our experiments, the
counting of MDN1 mRNA molecules per cell detected with
MCP-2xyeGFP revealed a mean Æ S.D. ¼ 10.8 Æ 6.1 mRNAs/
cell. Counting the MDN1 mRNAs per cell by smFISH gave
similar results, i.e., a mean Æ S.D. ¼ 9.5 Æ 4.4 mRNAs/cell
(Fig. 1h). These results show that live imaging faithfully reports
on MDN1 mRNA expression.
3.2 Visualizing
MS2-Labeled RNAs
in Mammalian Cells,
with a Focus
on Analyzing
Transcription
Dynamics
3.2.1 Lentiviral
Production of MCP-GFP
1. Seed HEK-293T cells at 4 Â 10
6 cells in 100 mm tissue culture
plate and incubate cells for 24 h.
2. Change HEK-293T cells into 5 mL of fresh medium without
antibiotics.
3. Transfect HEK-293T cells with JetPRIME (Polyplus transfection; according to the supplier’s recommendations) by preparing a mix of 10 μg DNA total with 8 μg of pHAGE-Ubc-NLSMCP-GFP, 0.4 μg of pHDM-Hgpm2, 0.4 μg of pHDM-tat1b,
0.4 μg of pRC-CMV-rev1b, 0.8 μg of pHDM-G, and 500 μL
jetPRIME buffer.
4. Mix by vortexing, add 20 μL of JetPRIME, vortex for 5 s, spin
down briefly, and incubate for 10 min at room temperature.
5. Add the transfection mix dropwise onto the HEK-293T cells.
Gently rock the plate back and forth and incubate at 37
C for
24 h (see Note 13).
6. Harvest medium containing lentivirus, filter it through a
0.45 μm filter into a sterile falcon tube, and keep it at 4
C.
7. Add 5 mL of fresh medium without antibiotics to the packaging cells and incubate at 37
C for 24 h for a new round of
lentivirus production.
8. Repeat steps 6 and 7 once. After the harvest of virus, discard
the HEK-293T cells with 10% bleach.
9. Pool the viral harvests of this and the previous days.
10. To concentrate lentiviral stocks, add Lenti-X Concentrator
(Clontech) to viral harvest and incubate at 4
C for 30 min to
overnight.
11. Centrifuge the mixture at low speed (1500 Â g for 45 min at
4
C) and discard the supernatant.
12. Resuspend the pellet in DMEM and aliquot for titration and
single-use aliquots. Store at À80
C (see Note 14).
Imaging Single mRNAs in Living Eukaryotic Cells
135
for FISH-quant analysis. 3D gaussian fitting of the single spots
generates a text file containing the x,y position, brightness, and
number of spots identified in each cell. In our experiments, the
counting of MDN1 mRNA molecules per cell detected with
MCP-2xyeGFP revealed a mean Æ S.D. ¼ 10.8 Æ 6.1 mRNAs/
cell. Counting the MDN1 mRNAs per cell by smFISH gave
similar results, i.e., a mean Æ S.D. ¼ 9.5 Æ 4.4 mRNAs/cell
(Fig. 1h). These results show that live imaging faithfully reports
on MDN1 mRNA expression.
3.2 Visualizing
MS2-Labeled RNAs
in Mammalian Cells,
with a Focus
on Analyzing
Transcription
Dynamics
3.2.1 Lentiviral
Production of MCP-GFP
1. Seed HEK-293T cells at 4 Â 10
6 cells in 100 mm tissue culture
plate and incubate cells for 24 h.
2. Change HEK-293T cells into 5 mL of fresh medium without
antibiotics.
3. Transfect HEK-293T cells with JetPRIME (Polyplus transfection; according to the supplier’s recommendations) by preparing a mix of 10 μg DNA total with 8 μg of pHAGE-Ubc-NLSMCP-GFP, 0.4 μg of pHDM-Hgpm2, 0.4 μg of pHDM-tat1b,
0.4 μg of pRC-CMV-rev1b, 0.8 μg of pHDM-G, and 500 μL
jetPRIME buffer.
4. Mix by vortexing, add 20 μL of JetPRIME, vortex for 5 s, spin
down briefly, and incubate for 10 min at room temperature.
5. Add the transfection mix dropwise onto the HEK-293T cells.
Gently rock the plate back and forth and incubate at 37
C for
24 h (see Note 13).
6. Harvest medium containing lentivirus, filter it through a
0.45 μm filter into a sterile falcon tube, and keep it at 4
C.
7. Add 5 mL of fresh medium without antibiotics to the packaging cells and incubate at 37
C for 24 h for a new round of
lentivirus production.
8. Repeat steps 6 and 7 once. After the harvest of virus, discard
the HEK-293T cells with 10% bleach.
9. Pool the viral harvests of this and the previous days.
10. To concentrate lentiviral stocks, add Lenti-X Concentrator
(Clontech) to viral harvest and incubate at 4
C for 30 min to
overnight.
11. Centrifuge the mixture at low speed (1500 Â g for 45 min at
4
C) and discard the supernatant.
12. Resuspend the pellet in DMEM and aliquot for titration and
single-use aliquots. Store at À80
C (see Note 14).
Imaging Single mRNAs in Living Eukaryotic Cells
135