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.

1. Engineering of Cells with Visible Plasmids
<ol>
	<li>Use standard restriction digestion or homologous recombination techniques to introduce a DNA fragment containing approximately 250 copies of the lactose operator sequence (LacO) into the plasmid to be visualized. Our plasmid was a BACmid of approximately 170 kbp and contained the entire genome of Kaposi's Sarcoma-associated Herpes Virus (KSHV) and encoded resistance to hygromycin 12.</li>
	<li>Introduce LacO-containing plasmid DNA into mammalian cells b...

<ol>
	<li>Cesarman, E., Chang, Y., Moore, P. S., Said, J. W., Knowles, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma-associated+herpesvirus-like+DNA+sequences+in+AIDS-related+body-cavity-based+lymphomas.">Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.</a> N. Engl. J. Med. 332, 1186-1191 (1995).</li><li>Chang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+herpesvirus-like+DNA+sequences+in+AIDS-associated+Kaposi's+sarcoma.">Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.</a> Science. 266, 1865-1869 (1994).</li><li>Knipe, D. M., Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology 2 volume set. , (2006).</li><li>Adams, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+latent+Epstein-Barr+virus+genomes+in+Raji+cells.">Replication of latent Epstein-Barr virus genomes in Raji cells.</a> J. Virol. 61, 1743-1746 (1987).</li><li>Humme, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EBV+nuclear+antigen+1+(EBNA1)+enhances+B+cell+immortalization+several+thousandfold.">The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 10989-10994 (1073).</li><li>Verma, S. C., Choudhuri, T., Robertson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimal+replicator+element+of+the+Kaposi's+sarcoma-associated+herpesvirus+terminal+repeat+supports+replication+in+a+semiconservative+and+cell-cycle-dependent+manner.">The minimal replicator element of the Kaposi's sarcoma-associated herpesvirus terminal repeat supports replication in a semiconservative and cell-cycle-dependent manner.</a> J. Virol. 81, 3402-3413 (2007).</li><li>Yates, J. L., Guan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus-derived+plasmids+replicate+only+once+per+cell+cycle+and+are+not+amplified+after+entry+into+cells.">Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells.</a> J. Virol. 65, 483-488 (1991).</li><li>Ye, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Kaposi's+sarcoma-associated+herpesvirus+latent+nuclear+antigen+leads+to+abortive+episome+persistence.">Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence.</a> J. Virol. 78, 11121-11129 (2004).</li><li>Robinett, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+localization+of+DNA+sequences+and+visualization+of+large-scale+chromatin+organization+using+lac+operator/repressor+recognition.">In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.</a> J. Cell Biol. 135, 1685-1700 (1996).</li><li>Nanbo, A., Sugden, A., Sugden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coupling+of+synthesis+and+partitioning+of+EBV's+plasmid+replicon+is+revealed+in+live+cells.">The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells.</a> Embo. J. 26, 4252-4262 (2007).</li><li>Herndier, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+Kaposi's+sarcoma+cell+line+that+induces+angiogenic+tumors+in+animals.">Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals.</a> Aids. 8, 575-581 (1994).</li><li>Zhou, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+infection+by+a+recombinant+Kaposi's+sarcoma-associated+herpesvirus+cloned+in+a+bacterial+artificial+chromosome:+application+for+genetic+analysis.">Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis.</a> J. Virol. 76, 6185-6196 (2002).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Pawley, J. B., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Points,+pixels,+and+gray+levels:+digitizing+image+data.">Points, pixels, and gray levels: digitizing image data.</a> Handbook of Biological Confocal Microscopy. , 59-79 (2006).</li><li>Cannell, M. B., McMorland, A., Soeller, C., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+enhancement+by+deconvolution.">Image enhancement by deconvolution.</a> Handbook of biological confocal microscopy. , 488-500 (2006).</li></ol>

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 cel...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent </td>
 <td>Company </td>
 <td>Catalogue number </td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>GIBCO</td>
 <td>11965</td>
 <td></td>
 </tr>
 <tr>
 <td>OptiMEM</td>
 <td>GIBCO</td>
 <td>31985</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Hyclone</td>
 <td>SH30910.03</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin</td>
 <td>Sigma</td>
 <td>P3032</td>
 <td></td>
 </tr>
 <tr>
 <td>Streptomycin sulfate</td>
 <td>Sigma</td>
 <td>S9137</td>
 <td></td>
 </tr>
 <tr>
 <td>Hygromycin</td>
 <td>Calbiochem</td>
 <td>400050</td>
 <td>400 &mu;g/ml for SLK; 300 &mu;g/ml for HeLa</td>
 </tr>
 <tr>
 <td>Isopropyl-b-D-thiogalactoside (IPTG)</td>
 <td>Roche</td>
 <td>10 724 815 001</td>
 <td>Final concentration 200 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Lipofectamine 2000</td>
 <td>Invitrogen</td>
 <td>11668-027</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>GIBCO</td>
 <td>15630</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass-bottom dishes</td>
 <td>MatTek</td>
 <td>P35G-1.5-10-C</td>
 <td></td>
 </tr>
 <tr>
 <td>CultFoil</td>
 <td>Pecon</td>
 <td>000000-1116-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Axiovert 200M</td>
 <td>Zeiss</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Colibri</td>
 <td>Zeiss</td>
 <td>423052-9500-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Neutral white LED</td>
 <td>Zeiss</td>
 <td>423052-9120-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Filter Cube 75 HE</td>
 <td>Zeiss</td>
 <td>489075-0000-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Plan-Apochromat 63x/1.4 objective</td>
 <td>Zeiss</td>
 <td>440762-9904-000</td>
 <td></td>
 </tr>
 <tr>
 <td>CascadeII:1024 EMCCD camera</td>
 <td>Photometrics</td>
 <td>B10C892007</td>
 <td></td>
 </tr>
 <tr>
 <td>Tempcontrol mini</td>
 <td>Zeiss/Pecon</td>
 <td>000000-1116-070</td>
 <td></td>
 </tr>
 <tr>
 <td>Tempcontrol 37-2 digital</td>
 <td>Zeiss/Pecon</td>
 <td>000000-1052-320</td>
 <td></td>
 </tr>
 <tr>
 <td>CTI Controller 3700 digital</td>
 <td>Zeiss/Pecon</td>
 <td>411856-9903</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective heater</td>
 <td>Zeiss/Pecon</td>
 <td>440760-0000-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Stage heating insert P</td>
 <td>Zeiss/Pecon</td>
 <td>411861-9901-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Stage-top Incubator S</td>
 <td>Zeiss/Pecon</td>
 <td>411860-9902-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Humidifier System</td>
 <td>Zeiss/Pecon</td>
 <td>000000-1116-065</td>
 <td></td>
 </tr>
 </tbody>
</table>

monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy

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.

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 cycl...

Watch this Scientific Journal Video about Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy at JoVE.com

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

monitoring-plasmid-replication-live-mammalian-cells-over-multiple

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JoVE Journal

Biology

11.7K visualizações.  University of Wisconsin - Madison. Um método de se observar as moléculas individuais de DNA em células vivas é descrito. A técnica baseia-se na ligação de uma proteína fluorescente etiquetado repressor lac para os locais de ligação para a engenharia de ADN de interesse. Este método pode ser adaptado para seguir muitas DNAs recombinantes em células vivas ao longo do tempo.