Department of Oncology, University of Wisconsin - Madison
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Norby, K., Chiu, Y. F., Sugden, B. Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy. J. Vis. Exp. (70), e4305, doi:10.3791/4305 (2012).
Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.
1. Engineering of Cells with Visible Plasmids
2. Development of the Imaging System
Our imaging system consists of a Zeiss Axiovert 200M equipped with a motorized stage and Plan-Aochromat 63x/1.4 oil objective. Fluorescence excitation light is provided by the "neutral white" LED of a Colibri system. Emission is detected by a CascadeII:1024 EMCCD camera. This is a cooled, back-thinned camera with a gain register for amplifying signals; the dark current is 0.061 electrons per pixel per second, and the quantum efficiency is greater than 0.90. The system is controlled by Axiovision 4.8 software, including the SmartExperiments module. For detection of tdTomato we use a Zeiss filter set 75HE for which 85% of the passed light can excite the fluor and 95% of the emitted light will pass the emission filter.
3. Visualization of Labeled DNA
4. Post-acquisition Processing of Images
Maximum intensity projections of z-stacks acquired in a representative experiment are shown in Figure 3. Typical plasmid signals are present in 3-8 slices of the z-stack, depending on whether they represent single or clusters of plasmids. In the case of EBV- and KSHV-derived plasmids, the signals move slowly within the cell until the cell reaches mitosis. The cells themselves migrate over the course of the experiment. They also become spherical and detach from the dish during mitosis. The cell cycle for healthy HeLa and SLK cells takes approximately 24 hr; cells of these types that take more than 30 hr to complete the cell cycle should be considered unhealthy. Daughter cells typically attach near each other and go on to divide at the same time in the next cell cycle. Only cells that can be shown to complete a cell cycle should be analyzed.
Figure 1. Scheme for making plasmids visible. (A) Tandem repeats of the lactose operator sequence (LacO) are cloned into the plasmid of interest. The lactose repressor protein (LacI) binds specifically to these sequences; fusion to a fluorescent protein recruits the fluorescent tag to the plasmid of interest. (B and C) The nucleus of a HeLa cell expressing LacI-tdTomato is fluorescent due to the nuclear localization signal on the fusion protein localizing it to the nucleus. KSHV genomes encoding LacO sequences recruit many copies of the fluorescent protein and stand out as bright signals over the background intensity of the nucleus in the absence of IPTG (B), while the LacI- fluorescent proteins are prevented to bind to its recognition LacO sequence in the presence of IPTG (C). These images are made computationally to include multiple z-planes in which the different signals reside.
Figure 2. Diagram of the plasmid visualization procedure. First, tandem LacO repeats are cloned into the plasmid of interest. That plasmid is introduced into cells by transfection. Clones of transfected cells are selected and screened, and then infected with a retrovirus expressing a fluorescent LacI fusion protein.
Figure 3. Review images to track plasmids throughout the cell cycle. Fluorescently tagged viral genomes in a HeLa cell (A) are synthesized in the S-phase (B) and partitioned to daughter cells during cell division (C-E) and can be followed for multiple generations. Shown are maximum intensity projections of z-stacks acquired of a HeLa cell cycle at five time points in a representative experiment. These images are made as in Figure 1B and C.
The method described here can be used to follow plasmids in live mammalian cells over time spanning multiple generations. By limiting exposure to excitation light, we have used these techniques to follow cells from newly-divided pairs through colonies of 16-32 cells, representing at least 72 hr and 3 cell divisions. These experiments provide information that cannot be obtained from static assays such as quantitative PCR, Southern blotting, and FISH.
It is essential to screen clones of cells for plasmids with a homogenous structure, as rearrangements can occur when using plasmids with repetitive sequences such as the tandem LacO repeats. In addition, it is useful to optimize retrovirus infection and screen clones of infected cells for an optimal signal intensity. Cells expressing too much LacI protein will have high background fluorescence in the nucleus, and those expressing too little will have weak signals. Finally, it is important to look at cell morphology and choose healthy cells when marking fields of view to use during their visualization.
This technique can be used to visualize nearly any DNA molecule, provided it can be engineered to include tandem repeats of the LacO sequence. Examples include cellular chromosomes, naturally occurring and artificial plasmids, and viral genomes packaged within virions. In experiments similar to that depicted in Figure 3, we found that our KSHV-derived plasmids appear to be partitioned randomly during cell division. A logical extension of the technique is to tag one DNA molecule as a LacO/LacI pair with one fluorescent protein, and another DNA molecule in the same cell as a Tetracycline Operator/Tetetracycline Repressor pair with another fluorescent protein. Experiments are limited by the amount of excitation light the cells can withstand before experiencing cytotoxic effects.
No conflicts of interest declared.
This work was funded by grants from the NIH and ACS, including T32CA009135, CA133027, CA070723, and CA022443. Bill Sugden is an American Cancer Society Research Professor.
|Fetal bovine serum||Hyclone||SH30910.03|
|Hygromycin||Calbiochem||400050||400 μg/ml for SLK; 300 μg/ml for HeLa|
|Isopropyl-b-D-thiogalactoside (IPTG)||Roche||10 724 815 001||Final concentration 200 μg/ml|
|Neutral white LED||Zeiss||423052-9120-000|
|Filter Cube 75 HE||Zeiss||489075-0000-000|
|Plan-Apochromat 63x/1.4 objective||Zeiss||440762-9904-000|
|CascadeII:1024 EMCCD camera||Photometrics||B10C892007|
|Tempcontrol 37-2 digital||Zeiss/Pecon||000000-1052-320|
|CTI Controller 3700 digital||Zeiss/Pecon||411856-9903|
|Stage heating insert P||Zeiss/Pecon||411861-9901-000|
|Stage-top Incubator S||Zeiss/Pecon||411860-9902-000|