The overall goal of the following experiment is to investigate the spatial organization of specific genomic loci in single cells in a robust fashion. This is achieved by first generating fluorescently labeled probes to ultimately bind and highlight the genomic region of interest. The next steps are to adhere, fix and perme cells of interest on a slide in order to permit probe access.
Then a simultaneous heat denaturation of probe and cellular DNA is followed by overnight probe hybridization wash steps to remove non-specifically bound probe and counter standing with dpi. To visualize the cell nucleus results obtained with fluorescence microscopy will ultimately reveal the nuclear position occupied by specific genomic regions. This method can help answer key questions in the nuclear organization field, such as determining functional interactions between genes.
The main advantage of this particular DNA FISH protocol is that it is fast, robust, and versatile. Although we demonstrate this method using E Es cells, it can also be applied to numerous other cell types, Directly labeled probes made from backs of 100 to 250. Kilobases consistently produce bright signals if smaller probes are required.
Use 40 to 50 kilobase smid, or even plasmids containing five to 10 kilobase inserts. Identify the back or smid clones corresponding to specific genes using the ensemble or UCSC genome browsers. To prepare high quality back DNA use either repeated precipitation or a commercially available kit, the cleanliness of the preparation is proportional to the volume of probe required.
Perform the labeling in two steps. First, use NIC translation to introduce amino allele, DUTP. Second, use chemical coupling to attach an AM mean reactive dye.
It is worth investing time in making bright probes as these are critical for strong fish signals. This procedure can be performed in parallel to the treatment of the cells and does not have to be done at the start of the day. First mixed 10 to 20 nanograms of directly labeled probe with six microliters of CO 1D NA for six micrograms.
Then add one microliter of single stranded DNA from salmon testes for 9.7 micrograms. Adjust the volume of the mix to 100 microliters with water. Next, add 10 microliters of three molar sodium acetate and 275 microliters of ethanol and mix by vortexing.
Allow the mixture to precipitate for at least one hour at minus 20 degrees Celsius. Next, spin the precipitation in a micro fuge at maximum speed for 30 minutes at four degrees Celsius. Then wash the pellet with 70%ethanol and spin it down again for five minutes at four degrees Celsius.
Next air dry the pellet. Then resus. Suspend it in five microliters of deionized form.
Amide at 37 degrees Celsius while shaking at 1000 RPM you foil to protect it from light. After 30 minutes, add five microliters of dextrin sulfate mix and continue the incubation with agitation. For 10 more minutes, The cells propensity to adhere to Slide greatly varies with cell type.
For each cell type, determine the optimum settling time and cell density empirically. For consistent results, cells should be in a single cell suspension To adhere mouse ES cells on a slide. Start by circling the area on the slide where the cells will be spotted.
Using a hydrophobic pen, have prepared a suspension of 20 to 50, 000 cells per 80 to 100 microliters in normal culture medium, add the cell suspension into the circle and allow the cells to attach for about three hours in a humidified incubator. After the slide attachment, fix the cells by gently submerging the slide in 4%PFA for 10 minutes. The slide should be flat in a tray like a tip box.
Using room temperature reagents in coplan jars. First quench the cells with tris chloride for 10 minutes. Second, permease the cells using a sapin and triton X 100 mix for 10 minutes.
Now wash the cells twice in PBS for five minutes per wash. Following the washes incubate the cells in 20%glycerol for at least 20 minutes. At this point, the slides can be stored in 50%glycerol at minus 20 degrees Celsius for several weeks.
Storage for a day or more should increase the signal strength. Be sure to equilibrate the cells to room temperature in 20%glycerol in PBS before proceeding. Now, freeze and thaw the slide three times using liquid nitrogen.
One slide at a time when the crackling sound stops. After a few seconds, remove the slide to a paper towel to defrost wait for the opaque frozen glycerol to disappear before freezing the slide again, up to 15 slides can be done comfortably in rotation for three freeze thaw cycles. Now wash the slides twice in PBS for five minutes per wash.
Then incubate the slides in 0.1 molar hydrochloric acid for 30 minutes. Follow the hydrochloric acid treatment with another five minute wash in PBS. Now perme ize the cells again with a stronger sapin and Triton X 100 mix for 30 minutes to wash the surfactants off the slides, use two five minute PBS washes.
The slides are now ready to equilibrate in 50%form amide into XSSC. This was should last at least 10 minutes while equilibrating retrieve the probe pipetted up and down a few times and then transfer 10 microliters per cell spot to a cover slip of the appropriate size. Remove a slide from the form amide and dry off any excess liquid around the cells with a paper towel, but do not allow the cell spot to dry out.
Now invert the cover. Slip onto the slide over the cells. Seal the cover slip to the slide with rubber cement.
Allow the cement to dry completely and keep the slide protected from light. Next, heat the slides to 78 degrees Celsius for precisely two minutes on a hot plate. This is critical to perform properly.
A cover must protect the cells from light during this step To preserve the nuclear morphology as much as possible. It is critical to not let the cells dry out and to ensure that the heat loc has the right temperature. Next place the slides in a light tight box with moist paper towels to provide humidity and incubate overnight at 37 degrees Celsius.
Begin this procedure by peeling off the rubber cement from around the cover slips. Then place each slide into X excess SC until the cover slips loosen and slide off. Next, wash the slides in 50%form amide in two x excess SC for 15 minutes at 45 degrees Celsius.
Keep a lid on the water bath to protect them from light. Then wash the slides in 0.2 XSSC for 15 minutes at 63 degrees Celsius. Then two XSSC for five minutes at 45 degrees Celsius, and finally to excess SE at room temperature for five minutes.
The remaining steps are all at room temperature, beginning with a five minute wash and PBS now stain the slides with dappy for two minutes and then detain the slides with PBS for five minutes. To mount the cover slip, apply 10 microliters of mounting medium per cell spot onto the right sized cover slip. Then dry the excess PBS from around the cell spot without letting the cells dry out.
Invert the cover, slip onto a slide and seal it with nail varnish using the fish probe preparation protocol. After NIC translation, a smear of DNA should be visible with the bulk of the fragments running between 150 and 700 base pairs. After chemical coupling, incorporation of the fluorescent dye can be gauged by spectroscopic analysis of the probe passing.
These quality control steps to judge the probe quality is critical by using such a probe. Automated imaging with an epi fluorescence microscope was feasible. This requires sharp, easily identifiable fish signals with little background.
This is a raw field of view image. The following images represent nuclei which have been identified and analyzed automatically. Various inter allelic and inter locus distances are shown top right and to either side below each nucleus.
In this enlargement, white spots represent the identified nuclei per field of view. These histograms show maximal inter allelic distances between green signals above and red signals below. All data are from P 20 germ cells with probes located on different chromosomes covering the HBB and HBA genes and a histone cluster.
These are examples of fetal liver nuclei stained with back probes covering mic and KC and Q1 genes. From these images, nuclear coordinates of fish signals can be obtained and spatial relations of genomic regions can be computed. Inter allelic distances are plotted as a fraction of the nucleus radius.
Red probes were significantly closer together than green probes in this data. Confocal microscopy, which requires more intense signals was also feasible. Image stacks were used for 3D modeling of fish signals within their chromosome territories.
These two probes in red and white are close to either end of mouse chromosome seven whole chromosome paint is shown in green. After watching this video, you should have a good understanding of how to perform DNA fish in order to investigate the spatial positioning of specific genomic loci in single cells. While attempting this procedure, it's important to remember that all treatment may affect nuclear architecture and it is important to include appropriate Following this procedure.
Other methods like immuno fish can be performed in order to answer additional questions like spatial relationships between genes and proteins.
