The overall goal of this procedure is to observe the partitioning of viral genomes in dividing cells. This is accomplished by first making a plasmid that can be visualized by fluorescence microscopy. Here a plasmid is engineered to include tandem repeats of the lactose operator sequence or LAC O.The second step is to introduce the plasmid into dividing cells, followed by infection of the cells with a retrovirus encoding the lactose repressor or lac eye fused to a fluorescent protein.
The lactose repressor fusion proteins will specifically bind the lactose operator sequences in the plasmid sub. The cells are monitored by fluorescence microscopy to track individual plasmids through multiple cell cycles. Ultimately, the images acquired are processed to determine how the viral genomes are maintained in dividing cells.
The main advantage of this technique over existing methods such as quantitative PCR Southern blotting or fluorescence in situ you hybridization, is that this technique allows individual plasmids to be observed at multiple points in time. To enable visualization of the plasmid by fluorescence microscopy, standard molecular cloning techniques are used to introduce A DNA fragment containing approximately 250 copies of the lactose operator sequence or LAC O into the plasmid. In this example, the plasmid is a back mid of approximately 170 kilobase pairs that contains the entire genome of cap's sarcoma associated herpes virus or KSHV and encodes resistance to hygromycin.
Introduce the LAC O containing plasmid DNA into hela and SLK mammalian cells by transfection In 60 millimeter dishes, incubate cells with 10 micrograms purified plasmid, DNA 25 microliters, lipectomy, 2000 0.5 milliliters, Optum, and 3.5 milliliters. DMEM for four hours. Change the medium to DMEM with 10%fetal bovine serum and allow the cells to recover for 48 hours.
Subsequently plate the transfected cells at low density in large dishes and culture for three weeks in DMEM with 10%fetal bovine serum and 300 micrograms per milliliter hygromycin to select for the introduced plasmid, use pieces of sterile trips and soaked filter paper to transfer hygromycin resistant colonies to new culture dishes. Place the dishes in the incubator after one to two weeks when transfected clones have grown to fill the dish isolate DNA for restriction digestion and southern blotting to ensure that the polymer of the LAC O plasmid is homogenous and of the expected size. This example blot shows that the LAC O polymer in clone one is homogenous and identical to that of the positive control.
In lane seven, if the plasmid has not been engineered to express a fusion of the lactose repressor lac eye infect suitable clones with a retrovirus encoding lac eye fused to a red fluorescent protein such as lac eye, TD tomato, TD tomato is chosen instead of proteins that fluoresce closer to green to minimize phototoxicity that would occur from multiple exposures over long times. Once the LAC eye fusion protein is introduced, culture, the cells in the presence of 200 micrograms per milliliter IPTG to block the fusion proteins from binding DNA. One of the most critical aspects of this procedure is obtaining clones with the appropriate fluorescence intensity.
A large number of clones should be screened to identify those with optimal fluorescence Culture. The cells for at least three days to allow expression of the LAC eye TD tomato protein screen clones for those with optimal levels of lac eye, TD tomato that reveal distinct signals with minimal background levels of unbound fusion protein, 24 to 48 hours before imaging the LAC I TD tomato expressing cells plate them in a 35 millimeter glass bottom dish at a density of less than 10%confluence. This will allow pairs of cells to divide into colonies of eight to 16 cells without overlapping one to three hours before the start of imaging.
Rinse the plate three times with culture medium that lacks both selective drugs and IPTG. This washes away non-viable cells and allows lac eye fusion proteins to bind LAC cytes in the plasmids. Replace the culture medium with two milliliters, DMEM with 10%fetal bovine serum and 25 millimolar Hippies incubate for one to three hours just before imaging replace the top of the culture dish with cult foil stretched in a mounting ring for a 35 millimeter dish.
This cover will inhibit evaporation of the culture medium, which would increase salt concentration over time and kill the cells. The imaging system used in this experiment consists of a Zeiss Axio 200 M inverted microscope, equipped with a motorized stage and an EM CCD camera for sensitive detection of signals. Fluorescent excitation light is provided by the neutral white LED of a Collibra system.
The imaging system is controlled by a software for automated acquisitions of images over longtime intervals with multiple Z stacks. A stage top incubator is used to maintain cells at 37 degrees Celsius, five to 10%carbon dioxide in a humidified chamber. Use a heated collar for the oil objective to prevent the objective from siphoning.
Heat from the sample allow sufficient time for the system to come to a stable temperature. To minimize movement artifacts during the experiment, the imaging system should sit on an air table to be isolated from vibrations. To begin the procedure for visualizing labeled DNA use DIC imaging to view the cells and determine the focal plane of the top and bottom of the cells in multiple fields of view in each field, move to a focal plane approximately one third of the way down from the top of the cell, save the x, y, Z coordinates in the list of saved stage positions.
Each of these saved positions will be the center of a Zack of images to capture the cell in the microscope control software, define a Zack of images to be performed at each saved stage position, define enough slices so that the entire cell will be captured along with extra slices above and below the planes in which the cells are located. Choose a Z step size of 0.35 to 0.50 microns to allow approximate nyquist sampling in the Z dimension. These slices allow detection following cell movements during the cell cycle and are also used in post-acquisition processing of the image stacks.
This will result in a stack of 40 to 60 images depending on the thickness of the cells. Define a time series experiment to collect a bright field image of the Zacks at 30 minute intervals and fluorescence images at 10 to 60 minute intervals. Do not use DIC imaging during the experiment as it requires more light than standard brightfield imaging, which can expose the cells unnecessarily to phototoxicity over the course of long experiments.
Start the software controlled experiment. Ensure that the system is not disturbed in way during the experiment. After all images for the experiment have been acquired, review the images for each Zack and time point crop out any unneeded areas of the images to reduce the computer processing time.
In the next step to reassign signal intensity to the pixel of origin in each Z stack. Use a deconvolution algorithm in Axio vision software based on the measured point spread function of the imaging system. The result of the deconvolution is shown here.
Examine the images to track the intensity changes and movements of plasmids during the cell cycle and the partitioning of plasmids to daughter cells. These images which are made computationally to include multiple Z planes in which the different signals reside, show that the nuclei of HELOC cells expressing LAC eye TD tomato are fluorescent due to the nuclear localization signal on the fusion protein, localizing it to the nucleus. In the absence of I-P-T-G-K-S-H-V genomes encoding LAC O sequences recruit many copies of the fluorescent protein and stand out as bright signals over the background intensity of the nucleus.
In contrast, in the presence of IPTG, the LAC eye fluorescent proteins are prevented from binding to its recognition lack O sequence, maximum intensity projections of Zack acquired of a he a cell cycle at five time. Points in a representative experiment are shown in this figure. The fluorescently tagged viral genomes in the cell visible in panel A are synthesized in the shown in panel B.The viral genomes are then partitioned to daughter cells during cell division as observed in panel C, D and E.The cell cycle for healthy HELOC cells takes approximately 24 hours, and daughter cells typically attach near each other and go on to divide at the same time in the next cell cycle.
Using this protocol, the viral genomes in the cell can be followed for multiple generations While attempting this procedure. It's important to remember to watch cell morphology in generation time to ensure the cells are healthy throughout the experiment.
