The overall goal of this molecular cytogenic method is to observe interchromosomal stable aberrations in the bone marrow cells of mice exposed to total body irradiation. This method can help answer the key question in the radiation biology field such as the risk of inducing stable chromosomal aberrations in the bone marrow cells of mice exposed to total body radiation. The main advantage of this technique is that it allows guided visualization of radiation induced stable genetic damages in the bone marrow cells of mice that can be propagated through many cell generations.
After isolating bone marrow from mouse femur and tibia bones and making a single cell suspension according to the text protocol, carefully overlay the cell suspension on an equal volume of bone marrow mononuclear cell separation medium. Centrifuge the gradient at 400 times G at room temperature for 30 minutes. Then, carefully collect the buffy coat without disturbing the remaining layers and transfer the solution into a new 15 milliliter centrifuge tube.
To prepare metaphase cell spreads, add 10 milliliters of PBS to the tube containing the buffy coat. Then centrifuge the tube at 400 times G at room temperature for five minutes. Next, carefully remove the supernatant and gently tap the tube to break up the cell pellet.
Then add 10 milliliters of PBS and centrifuge the suspension again. Remove the supernatant without disturbing the cell pellet. Break up the pellet and add drop by drop four milliliters of pre-warmed hypotonic 0.075 molar potassium chloride solution with gentle, constant shaking.
Incubate the cell suspension at 37 degrees Celsius for 20 minutes in a water bath. Then, add an equal volume of fixative to the tube and mix gently by inverting the tube. Centrifuge the tube, and remove the supernatant.
Then, break up the cell pellet and add fresh fixative. Repeat the spinning and the addition of fixative five more times. After resuspending the cells in 400 to 600 microliters of fixative, drop 30 microliters of fixed cells onto precleaned and wet slides tilted at a 45 degree angle and allow the slides to air dry completely overnight.
To carry out mouse chromosome painting by mFISH place the slide containing the metaphase cell spreads in 2X SSC for two minutes at room temperature. Then, dehydrate the slide by serial ethanol washing in 70%80%and 100%ethanol for two minutes in each wash. Preheat 40 milliliters of denaturation solution in a glass coplin jar to 70 degrees Celsius for 30 minutes.
Then transfer the slide into the prewarmed solution and incubate the sample for one to 1 1/2 minutes to denature the chromosomes. Immediately use ice cold 70%ethanol to quench the slide for two minutes to stop the denaturation process and prevent denatured chromosomes from reannealing. Then dehydrate the slide using an ethanol series beginning with 80%ethanol for two minutes.
Next, place the slide in 100%ethanol for two minutes. Then completely dry the slide at room temperature. To denature the probe, briefly centrifuge the probe mixture supplied by the manufacturer, then transfer 10 microliters into a 500 microliter snap cap centrifuge tube and incubate the tube in a water bath at 80 degrees Celsius for seven minutes.
Now place the tube containing the denatured probe into a water bath at 37 degrees Celsius for 10 minutes. Then apply the probe mixture onto a slide with denatured chromosomes. Carefully cover the area with an 18 millimeter by 18 millimeter glass coverslip and eliminate any visible air bubbles by very gently pressing the coverslip to the slide.
Use rubber cement to seal all four sides of the coverslip. And incubate the slide in the dark in a humidified chamber at 37 degrees Celsius for 12 to 16 hours. Following hybridization, carefully remove the rubber cement and coverslip and place the slide in prewarmed 0.4X SSC at 74 degrees Celsius for five minutes.
Then transfer the slide into washing solution three for two minutes. Add 20 microliters of antifade mounting medium with DAPI counterstain onto the slide and cover it with a glass coverslip. Use lab tissue paper to gently press on the coverslip to remove any air bubbles and excess mounting solution.
Then use nail polish to seal the edges of the coverslip. View the slides using a fluorescent microscope equipped with the appropriate filters. For spectral karyotyping of mouse chromosomes, after the probes are hybridized onto the denatured chromosomes and the samples are washed according to the text protocol, apply 80 microliters of SY5 staining reagent to the slide.
Place a 24 by 60 millimeter plastic coverslip on top of the sample, and incubate it in the dark in a humidified chamber at 37 degrees Celsius for 40 minutes. Dip the slide into a glass coplin jar containing prewarmed washing solution three and incubate it in a water bath at 45 degrees Celsius with shaking for two minutes. Repeat the wash three times.
Apply 80 microliters of SY5.5 staining reagent to the sample, cover it with a 24 by 60 millimeter plastic coverslip, and incubate it in the dark in a humidified chamber at 37 degrees Celsius for 40 minutes. Use prewarmed washing solution three to wash the slide three times then hold the slide in a tilted position against a paper towel to drain the excess fluid. Add 20 microliters of antifade DAPI reagent to the sample and carefully place a glass coverslip on top without introducing any air bubbles.
Use nail polish to seal the edges and observe the sample with an epifluorescence microscope equipped for capturing sky images. This figure shows representative metaphase cell spreads with normal and aberrant mouse chromosome pairs one, two, and three. Here is a stable aberration involving chromosome one where a part of chromosome one has been integrated into a non-painted DAPI chromosome while another part has formed an acentric fragment.
In this example, a part of chromosome two has been integrated into a non-painted chromosome. This stable aberration involves chromosome three. Stable aberrations involving all three painted chromosomes are seen in this metaphase spread.
Shown here is an example of spectro karyotyping of a normal female mouse. This panel represents a stable aberration involving chromosomes four and 12. Finally, the stable chromosomal aberration in this panel involves chromosome seven and 10.
Once mastered, this technique can be done within 48 to 72 hours if it is done properly. While attempting this procedure, it is important to remember that only proliferating cells should be used. Following this procedure and other methods like in band can be used to answer additional questions like whether inter-chromosomal stable aberrations are present in the irradiated cells.
After its development, this technique paved the way for the researchers in the field of molecular cytogenetics to explore the association between genetic aberrations with the various diseases. After watching this video you should have a clearer idea how to determine inter-chromosomal stable aberrations. Don't forget that working with colchicine can be extremely dangerous because it is carcinogen and precautions such as wearing gloves should always be taken while performing this experiment.
