3.2.2 Generating Stable
Cell Lines Expressing
MCP-GFP in MS2x64 RNA
Reporter Cell Line
1. Seed MS2x64 RNA reporter cells (see Note 4) to a low density
(5000 cells per well) in a 12-well plate.
2. Thaw a lentiviral single-use aliquot at 37
C, prepare a range of
virus dilutions (e.g., 1:10; 1:50; 1:100) in DMEM (the volume
of 300 μL is enough to cover a 12 well-plate dish) without
serum and 6 μg/mL of polybrene (see Note 15), and mix well.
3. Remove the medium from cells, rinse once with DMEM,
remove and add the virus dilution, and incubate at 37
C.
Tilt the vessel back and forth to mix the virus every 20 min.
After 2 h, add 1 mL of fresh medium with serum and allow cells
to recover overnight.
4. Change medium the next day and treat all culture supernatant
as hazardous waste for several days afterward (5 days). Expand
and passage cells as normal for a week.
3.2.3 Screening
Polyclonal Population
Expressing MCP-GFP
1. Grow a fraction of the cells on 22 Â 22 mm coverslips in a
6-well plate dish.
2. Wash briefly in 1Â PBS and fix the cells with PFA 4% in PBS for
20 min at room temperature.
3. Wash briefly in 1Â PBS, add 20 μL of Vectashield mounting
medium on glass microscopy slides, and mount the coverslips
upside down.
4. Using an epifluorescence microscope, select the pool of cells
based on GFP expression (see Note 16).
3.2.4 Growing Cells
for Live Imaging
1. Split the cells to 50% confluence 1 day before imaging on a
25 mm diameter coverslips or a glass-bottomed tissue culture
plates (based on the microscope used).
2. Set up the incubator chamber of the microscope at 37
C and
5% CO 2 1 h before starting your experiment to avoid thermal
fluctuation leading to optical instability and cellular stress.
3. Set up the coverslips on the microscopic chamber and 30 min
before starting imaging replace the media with live-cell imaging
medium supplemented with 10% FBS and P/S.
3.2.5 Live Image
Acquisition
1. Determine experimental parameters for live-cell imaging. To
minimize photobleaching, the light intensity and the exposure
time need to be set to the lowest values allowing visualization
of single pre-mRNA molecules in the nucleus. These parameters are dependent on the microscope used (see Note 6)
and the light source and should be strictly determined when
starting imaging (see Note 17). This should be done at the
beginning of each experiment.
2. For each cell, determine the correct focal plane, as well as the
boundaries of the Z-stacks (see Note 18). For transcriptional
studies, two types of movies can be recorded: short movies (fast
136
Xavier Pichon et al.
Cell Lines Expressing
MCP-GFP in MS2x64 RNA
Reporter Cell Line
1. Seed MS2x64 RNA reporter cells (see Note 4) to a low density
(5000 cells per well) in a 12-well plate.
2. Thaw a lentiviral single-use aliquot at 37
C, prepare a range of
virus dilutions (e.g., 1:10; 1:50; 1:100) in DMEM (the volume
of 300 μL is enough to cover a 12 well-plate dish) without
serum and 6 μg/mL of polybrene (see Note 15), and mix well.
3. Remove the medium from cells, rinse once with DMEM,
remove and add the virus dilution, and incubate at 37
C.
Tilt the vessel back and forth to mix the virus every 20 min.
After 2 h, add 1 mL of fresh medium with serum and allow cells
to recover overnight.
4. Change medium the next day and treat all culture supernatant
as hazardous waste for several days afterward (5 days). Expand
and passage cells as normal for a week.
3.2.3 Screening
Polyclonal Population
Expressing MCP-GFP
1. Grow a fraction of the cells on 22 Â 22 mm coverslips in a
6-well plate dish.
2. Wash briefly in 1Â PBS and fix the cells with PFA 4% in PBS for
20 min at room temperature.
3. Wash briefly in 1Â PBS, add 20 μL of Vectashield mounting
medium on glass microscopy slides, and mount the coverslips
upside down.
4. Using an epifluorescence microscope, select the pool of cells
based on GFP expression (see Note 16).
3.2.4 Growing Cells
for Live Imaging
1. Split the cells to 50% confluence 1 day before imaging on a
25 mm diameter coverslips or a glass-bottomed tissue culture
plates (based on the microscope used).
2. Set up the incubator chamber of the microscope at 37
C and
5% CO 2 1 h before starting your experiment to avoid thermal
fluctuation leading to optical instability and cellular stress.
3. Set up the coverslips on the microscopic chamber and 30 min
before starting imaging replace the media with live-cell imaging
medium supplemented with 10% FBS and P/S.
3.2.5 Live Image
Acquisition
1. Determine experimental parameters for live-cell imaging. To
minimize photobleaching, the light intensity and the exposure
time need to be set to the lowest values allowing visualization
of single pre-mRNA molecules in the nucleus. These parameters are dependent on the microscope used (see Note 6)
and the light source and should be strictly determined when
starting imaging (see Note 17). This should be done at the
beginning of each experiment.
2. For each cell, determine the correct focal plane, as well as the
boundaries of the Z-stacks (see Note 18). For transcriptional
studies, two types of movies can be recorded: short movies (fast
136
Xavier Pichon et al.