Flag antibody. One clone was further characterized and used
for the following experiments. Isogenic stable cell lines expressing the HIV-1 MS2x64 reporter gene were created using the
Flp-in system in a HeLa H9 AAVS1-Tat cell line [60]. Flp-in
integrants were selected on hygromycin (150 μg/mL). The
MS2 tag is located in the intron of the reporter and thus labels
only the pre-mRNA. Note that splicing of this reporter occurs
post-transcriptionally and is not disturbed by the MS2 repeat
[60]. We found that this setup was the most appropriate to
visualize transcription because it allows the use of large tags
without compromising the mRNA fate. Individual clones were
picked and analyzed by in situ hybridization. One clone was
further characterized and used for the following experiments.
5. A ZEISS Axioimager Z1 wide-field microscope equipped with
a Plan Apochromat 63Â objective, N.A. 1.4 oil-immersion
objective (ZEISS), was used with a ZEISS VSG HBO
100/001.26E illuminating System, and Zyla 4.2 sCMOS
Camera (2048 Â 2048 pixels; 6.5 μm pixel size, from Andor).
We acquire data using 21 optical sections with a z-step size of
0.3 μm. MetaMorph (Molecular Devices) software is used for
instrument control as well as image acquisition.
6. An inverted OMX Deltavision microscope in time-lapse mode
with temperature-controlled chamber with CO 2 , together with
a x100, N.A. 1.4 objective and EMCCD cameras Evolve
512 Â 512, was used for live-cell imaging. Spinning disk confocal or HiLo microscopes equipped with 60Â or 100Â objectives with a N.A. >1.3 are also suitable.
7. To maximize the brightness of the tagged mRNAs, we recommend tagging the mRNA of interest with 24xMBSV6. However, as we discussed in previous publications [43, 58], for
mRNAs that are strongly expressed and that have a short halflife (i.e., GAL1 mRNA), tagging with 24 stem-loops can be
suboptimal. This is because when many mRNAs have to be
rapidly degraded, the presence of 24 MS2 loops, even if they
are low-affinity variant, can cause a delay in the degradation of
the MS2 array. In this case the mRNAs can be tagged with
12xMBSV6. We recommend always testing whether the insertion of the MS2 loops affects the stability, the localization, and
the expression of the mRNA of interest by comparing the
tagged mRNA (with or without the expression of MCP) to
the untagged mRNA by smFISH [43, 58].
8. Transformed cells can be frozen at this stage. Glycerol stocks
can be prepared by mixing 1 mL of exponentially growing
culture with 1 mL of 60% glycerol in YPD. Mix thoroughly
and freeze in cryo-tubes at À80
C. We did not notice a
reduction in live imaging quality if the cells are thawed instead
of using fresh transformations.
138
Xavier Pichon et al.
for the following experiments. Isogenic stable cell lines expressing the HIV-1 MS2x64 reporter gene were created using the
Flp-in system in a HeLa H9 AAVS1-Tat cell line [60]. Flp-in
integrants were selected on hygromycin (150 μg/mL). The
MS2 tag is located in the intron of the reporter and thus labels
only the pre-mRNA. Note that splicing of this reporter occurs
post-transcriptionally and is not disturbed by the MS2 repeat
[60]. We found that this setup was the most appropriate to
visualize transcription because it allows the use of large tags
without compromising the mRNA fate. Individual clones were
picked and analyzed by in situ hybridization. One clone was
further characterized and used for the following experiments.
5. A ZEISS Axioimager Z1 wide-field microscope equipped with
a Plan Apochromat 63Â objective, N.A. 1.4 oil-immersion
objective (ZEISS), was used with a ZEISS VSG HBO
100/001.26E illuminating System, and Zyla 4.2 sCMOS
Camera (2048 Â 2048 pixels; 6.5 μm pixel size, from Andor).
We acquire data using 21 optical sections with a z-step size of
0.3 μm. MetaMorph (Molecular Devices) software is used for
instrument control as well as image acquisition.
6. An inverted OMX Deltavision microscope in time-lapse mode
with temperature-controlled chamber with CO 2 , together with
a x100, N.A. 1.4 objective and EMCCD cameras Evolve
512 Â 512, was used for live-cell imaging. Spinning disk confocal or HiLo microscopes equipped with 60Â or 100Â objectives with a N.A. >1.3 are also suitable.
7. To maximize the brightness of the tagged mRNAs, we recommend tagging the mRNA of interest with 24xMBSV6. However, as we discussed in previous publications [43, 58], for
mRNAs that are strongly expressed and that have a short halflife (i.e., GAL1 mRNA), tagging with 24 stem-loops can be
suboptimal. This is because when many mRNAs have to be
rapidly degraded, the presence of 24 MS2 loops, even if they
are low-affinity variant, can cause a delay in the degradation of
the MS2 array. In this case the mRNAs can be tagged with
12xMBSV6. We recommend always testing whether the insertion of the MS2 loops affects the stability, the localization, and
the expression of the mRNA of interest by comparing the
tagged mRNA (with or without the expression of MCP) to
the untagged mRNA by smFISH [43, 58].
8. Transformed cells can be frozen at this stage. Glycerol stocks
can be prepared by mixing 1 mL of exponentially growing
culture with 1 mL of 60% glycerol in YPD. Mix thoroughly
and freeze in cryo-tubes at À80
C. We did not notice a
reduction in live imaging quality if the cells are thawed instead
of using fresh transformations.
138
Xavier Pichon et al.