The aim of this procedure is to use the fluorescence in situ two hybridization technique to determine the sex chromosome or translocation status of in vitro human embryos at risk of genetic abnormality. This is accomplished by lysing, a single cell biopsied from a day three embryo onto a glass slide and air drying the nucleus. The second step is to code D nature and hybridize the target nucleus DNA with DNA probes labeled with fluorochromes of different colors.
Next excess probe is removed using a stringent wash and the nucleus is stained with a DNA specific fluorescent stain. The final step is to view the nucleus using a fluorescence microscope and to capture digital images. Ultimately, results can be obtained that show the copy number of the targeted chromosome regions through fluorescence microscopy.
Generally, individuals new to this technique will struggle because it's at the very limit of the fluorescence in situ hybridization technique. 24 hours in advance, prepare the cell lysis buffer for spreading cells. Filter the solution and dispense one milliliter aliquots into 10 to 15, 1.7 milliliter sterile micro centrifuge tubes close and label the tubes prior to freezing at minus 20 degrees Celsius just before the biopsy procedure.
Defrost the lysis buffer at room temperature. Score a small circle approximately five millimeters in diameter on the underside of an amine coated slide using a diamond pen, and then using a hard four H pencil pre-labeled the slide BLO with a latex glove. To remove any graphite dust, use a separate slide for each blaster mirror in numerical order.
Now place a small volume of lysis buffer within the circle. Transfer the biopsy blaster into the lysis buffer. Add lysis buffer as necessary to lyse the cell.
Observe the nucleus to ensure that it remains within the circle and is not lost. If the cell does not have a nucleus or has multiple nuclei, biopsy another cell. Leave the slide to air dry at room temperature.
First, pre-wash the samples in coplan jars using phosphate buffered saline for five minutes at room temperature. Then rinse twice in sterile distilled water dehydrate with an ethanol series for two minutes each at room temperature and air dry. Ensure slides are fully immersed and if any graphite dust floats to the surface, soak up with a clean tissue.
Record the position of the nucleus within the circle by visualizing with a phase contrast microscope. Then dehydrate the samples with 100%ethanol for two minutes at room temperature and air dry. Now apply two microliters of probe mixture and cover with a nine by nine millimeter cover slip.
Seal the edges of the cover slip with rubber cement. Perform the coded naturing. Step on a heat block at 75 degrees Celsius for five minutes, and then hybridize overnight in a humidified chamber at 37 degrees Celsius for each coupling jar.
Equilibrate 50 milliliters of 0.4 times SSC stringent wash solution in a 71 degree Celsius water bath. Place the stringent wash bottle and the empty coupling jar in the water bath at room temperature heat to 71 degrees Celsius pH the stringent wash and decant into cochin jars. Proceed to carefully remove the rubber cement from each slide and rinse off the cover slip using four times SSC 0.05%tween 20 pH seven at room temperature.
Wash the samples in the 0.4 times SSC stringent wash at 71 degrees Celsius for five minutes, followed by four times SSC 0.05%Tween 20 at room temperature for two minutes. For probes that are indirectly labeled, drain the slides of excess liquid and apply 20 microliters of fluorescently conjugated antibody under a 20 by 20 millimeter square of paraform. Incubate in a humidified chamber at 37 degrees Celsius for 15 minutes.
Remove the para film and perform the following washes at room temperature for two minutes. Once in four times SSC 0.05%tween 20 and twice for two minutes in PBS. After draining the slides, apply six microliters of vector shield mounting medium containing DPI to a 22 by 22 millimeter.
Number zero cover slip and invert the slide over the cover. Slip blot and seal the edges of the cover slip with clear nail varnish. Finally, analyze the samples by fluorescence microscopy.
Score each nucleus by two independent analysts. Also use imaging software to capture an image of the nucleus for confirmation of the visual diagnosis and for image archiving as part of laboratory quality assurance. Here a metaphase spread and an interphase nucleus prepared from peripheral blood lymphocytes indicate a carrier of a reciprocal translocation between the short arms of chromosomes five and nine with break points at five P 14.3 and nine P two, 4.1.
Fish probes illuminate both trans located segments and the centric segment of chromosome nine signals. In interface blast mere nuclei from day three, embryos can be captured and then merged to form a composite image. Two signals for each probe indicate two copies of each locus, which is consistent with a normal or balanced complement.
For the translocation chromosomes colored signals indicate one copy of the trans located segment of chromosome five three copies of the trans located segment of chromosome nine and two copies of the centric segment of CH chromosome nine. This data is consistent with an unbalanced product of man somi for five P 14.3 to five PT and trisomy for nine P two, 4.1 to nine pt. After watching this video, you should have a good understanding of how to analyze a single cell biopsied from a human embryo and prepare a single nucleus using the fluorescence in situ hybridization technique.
This is important to achieve reproducible and optimum test results to determine sex chromosome complement, or translocation status of an embryo.
