Authors: Yuri G. Strukov and Andrew S. Belmont
This protocol was adapted from “Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes,” Chapter 25, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.
The discovery and use of fluorescent proteins to label chromosomal proteins has yielded basic structural information as well as insights into dynamics that were previously inaccessible. This protocol describes a method for tagging specific chromosome sites using the lac operator/repressor system, wherein direct repeats of bacterial operator sequences are coupled with green fluorescent protein (GFP)-tagged proteins that recognize these sequences. Direct lac operator repeats are generated by directional cloning. Although the use of direct repeats, as opposed to inverted repeats, reduces recombination within the bacterial host at high copy number, even the direct repeats are unstable, requiring the use of special bacterial hosts and low-copy-number plasmids for cloning. The introduction of the lac operator repeats into eukaryotic cells uses traditional transformation methods. Techniques for the isolation of stable cell clones with varying transgene copy numbers are described in the protocol, as are several methods for visual screening of large numbers of stable cell clones to isolate rare clones containing labeled chromosomal regions with desired features.
In principle, chromosome tagging can be accomplished through the use of any repetitive sequence to which a specific protein binds. The first application of this approach used the bacterial lac operator and Lac repressor combination. The lac operator/Lac repressor-labeling system has been successfully adapted for tagging chromatin in live bacteria, yeast, and mammalian tissue culture cells (Fig. 1), as well as in multicellular organisms including Caenorhabditis elegans, Drosophila, and Arabidopsis. Subsequent approaches have used tet operator or glucocorticoid receptor element repeats. Recently, the lac operator system has been used in combination with the bacteriophage viral replicase translational operator and several color variants of GFP to label DNA, RNA, and protein in living cells.
Figure 1. Examples of different stable cell lines with lac operator-tagged chromosome regions visualized by GFP-Lac repressor in vivo expression. (Top) Three different gene-amplified cell lines, derived from multicopy plasmid insertions, each with different characteristic conformations of the lac operator-tagged chromosome region. (Top, left) A03 cells with a heterochromatic-like amplified chromosome region. A 90-Mb region forms an ~1-μm diameter spot during most of interphase, not much larger than its ~1.3-μm metaphase chromosome length. (Top, middle) PDC cell line in which the amplified chromosome region appears as coiled, large-scale chromatin fibers by light and electron microscopy. (Top, right) Bb cell line in which extended large-scale chromatin fibers are observed. In this example, what appear to be two sister chromatids are shown. (Bottom) Different stages of gene amplification. (Bottom, left) EP1-4 cell line containing a single-copy insertion of plasmid transgene containing lac operator direct repeats. (Bottom, middle and bottom, right) Two different stages of gene amplification using methotrexate selection. Chromosome regions consisting of clusters of individual dots are observed. These increase in number with increased amplification between bottom, middle, and bottom, right. Bars (top), 1 μm; (bottom), 2 μm.
Preparing Vector DNA Containing Large Direct Repeats
Making Stable Cell Lines Containing lac Operator Repeats
3.Grow cells for transfection for ~1 wk.
4.Purify plasmid DNA for transfection as follows:
5.Transform cells using calcium-phosphate-precipitated DNA.
Use only chemical transformation with competent cells, not electroporation, because increased recombination following electroporation has been observed. The efficiency of transfection using calcium-phosphate-precipitated DNA strongly depends on the growth state of recipient cells, concentration of calcium and phosphates, concentration of DNA, pH, temperature, and duration of the precipitation reaction. It is advisable to prepare several buffers with slightly different pH values (see Step5.iii below).
Enrichment of Stable Cells with Large-Copy-Number Inserts Using Fluorescence-Activated Cell Sorting
This procedure describes the selection of CHO cells expressing high amounts of exogenous DHFR from a pool of stably transformed cells using cell sorting, a strategy to enrich for cells harboring large chromosome insertions of the DHFR-containing transgene. Selection is based on binding a fluorescent cell-permeable DHFR inhibitor to the pool of cellular DHFR. Cells with high fluorescence show a high total expression of DHFR, which correlates roughly with copy number (as well as chromosome position effects).
6.Select and sort the cells as follows: - i. Grow cells in 25-cm2 tissue culture flasks until they are in log phase. - ii. Replace the growth medium with fresh F-12 (HAM) medium supplemented with FBS (10%) and FMTX (20 μM). Add glycine (100 μM), hypoxanthine (30 μM), and thymine (30 μM) to the medium to relieve the toxic effects of FMTX. Incubate the cells for 8 h. - iii. Harvest cells by trypsinization, and keep them in sterile PBS on ice. - Because FMTX is retained in cells at concentrations high enough to cause damage, cells are viable in PBS on ice only for several hours. - iv. Sort the cells into flasks or 96-well plates. - v. Grow enough cells for freezing, and freeze them after screening.
7.Prepare a large number of clones for screening or subcloning as follows:
8.If visual screening of the clones is unnecessary, then pick and harvest a large number of colonies from the 150-mm-diameter Petri dish (from Step 7), using one of the subcloning methods described below. If live visual screening is desired, refer to the Discussion regarding subcloning stable transformants.
Trypsin Method for Picking Colonies
Micropipette Tip Method for Picking Colonies
This method works well with more densely packed colonies, and it is faster than the trypsin method (Steps 8.i-8.ix). A few hundred colonies can be easily transferred within a couple of hours. Once this method is optimized, efficiency is near 100%, although the number of cells transferred per colony into the well may be somewhat lower.
In general, screening of clones uses either a visual, microscope-based screen (if possible) or a molecular or biochemical screen. In some cases, both might be combined, either as two sequential screens or one parallel screen. An example from our work is a screen of a set of 96 subclones transfected with a nonfluorescent VP16 transcriptional activator fusion protein. A visual immunofluorescence assay was used to test directly for expression of the activator, and a transient transfection expression assay was run to test the capability of each clone to activate a luciferase reporter gene. The intersection of these two screens yielded a clone that induced chromosome repositioning after induction of VP16 targeting.
A starting point for subcloning is to determine roughly what fraction of stable transformants show the desired properties. If this fraction is large, then we typically carry out the initial transformation in a single flask, followed by subsequent subcloning of the mixed stable transformant pool by serial dilution cloning into 96-well plates. A range of dilutions should be used to ensure success. The advantage of serial dilution is that the investigator can more easily verify whether the cells in a given well actually arise from a single clone. If the fraction of desired cells is small (a few percent or less), and if a visual screen is possible, we prepare a single coverslip from the mixed pool of stable transformants and obtain an initial estimate of exactly how many clones need to be screened. One approach is to initially subclone in pools of clones (e.g., five to 10 per well). In theory, this should allow cloning of even rare transformants through several stages of subcloning. Often, however, the desired subclones are rare because of a selective bias against growth of these clones. In this case, if the pool size is too great, the percentage of desired cells can drop in the culture prior to the next stage of subcloning, producing diminishing returns. A better approach, where possible, is cell sorting with flow cytometry to enrich the cell population with cells containing the desired expression levels (see Step 6 above). When large numbers of clones are required, and if visual screening is unnecessary, we simply pick large numbers of colonies from a 150-mm-diameter Petri dish, using either small, trypsin-soaked pieces of filter paper (Steps 8.i-8.ix above) or micropipette tips (Steps 8.x-8.xiv above). These are used to deposit clonal cells into 96-well plates. Both methods are very rapid but have a high risk of clone contamination, due to the use of a single Petri dish. Any clones selected, based on a secondary screen, must go through an additional round of clone “purification” using serial dilution.
Live visual screening is a little more complicated. An easy, low-cost method is to cover a large Petri dish (i.e., 60-mm diameter) with a layer of small, round coverslips (12-mm diameter). Cells are plated at low density such that one to five colonies grow per coverslip. Under sterile conditions, the coverslips are transferred to a Petri dish containing a hole over which a large glass coverslip is attached with vacuum grease and transported to the microscope for visual screening. Each colony on a given coverslip is screened. If a positive clone is found, then the coverslip is recovered under sterile conditions, and the region of the coverslip containing the desired colony is broken off with a diamond pen and passaged. A more convenient, but more expensive, method is to pick up a large number of colonies, as described above, and place them into a 96-well, glass-covered microtiter dish (Whatman, Polyfiltronics). These dishes are available with number 1-1/2 thickness coverslips suitable for high-resolution imaging. After the cells in each well grow to a sufficient density, the 96-well plate can be directly screened using an inverted microscope. These plates, however, are expensive (about $25 per plate at the time of publication) and in high demand; thus they may be back-ordered for 3-6 mo at a time. Also, significant variability exists between batches of plates in their ability to support cloning at high efficiency. This appears to be related to contamination of the plates with residual glue. Our experience is that many lots of these plates support cell growth at high density but not at the limiting dilutions required for cloning.
It is important to formulate a general approach to determine whether the biological phenomenon under study can be successfully recapitulated during biological imaging. In our experience, phototoxicity remains a serious problem. This is especially true for experiments that exploit the labeling of small, selected chromosome regions, where high magnification, low signal intensity, and correct focus require significant photoexposure. Different biological phenomena show very different thresholds for phototoxicity. Whereas chromosome decondensation after mitosis or rapid constrained chromosome motions appear to be relatively photoinsensitive, long-range chromosome movements induced by targeting of VP16 are extremely photosensitive, and VP16-induced large-scale chromatin decondensation is somewhere in between. Thus, most live-cell imaging studies should begin with a careful statistical survey using a fixed-cell approach. The goal is to define a biological phenomenon using standard cytological approaches and generate statistics that can be used to monitor reproducibility in a corresponding live-cell imaging experiment. If the statistics are not reproducible, then there is a problem with either the conditions used to grow the cells on the microscope stage or phototoxicity. In the latter case, the statistical approach can be used to determine the maximum photoexposure permitted that still allows “the biology” to proceed normally. If the phenomenon cannot be studied with fixed samples, for example, characterization of chromatin dynamics, then different approaches must be taken. At a minimum, we would like to demonstrate that the phenomenon being measured does not vary as a function of light exposure or time the cells spend being grown and imaged on the microscope stage, assuming that no time dependence is expected.
This work was supported by National Institutes of Health grants GM-42516 and GM-58460 to A.B.