The overall goal of this procedure is to assess ploy of metaphase two eggs After experimental manipulation begin by collecting fully grown mouse cytes from primed to sexually mature females. Then micro inject the oocytes with materials of choice to perturb the protein of interest and mature cytes in vitro to metaphase of meiosis two. Next stimulate chromosome spreading in the egg by treatment with mantro, which results in a monopolar spindle.
Ultimately, these results can depict ploy status of the egg through immunofluorescence and confocal microscopy. This method can help answer key questions in the reproductive biology and chromosome segregation fields such as understanding the molecular mechanisms that lead to an fluidity Visual demonstration of this method is critical as the micro injection technique is difficult to learn because several steps are hard to convey In writing. Intraperitoneal inject sexually mature female mice with five IUs of pregnant marere serum gonadotropin to maximize the number of antral follicles isolated from each mouse.
Approximately 48 hours after PMSG priming warm three milliliters per mouse of collection medium at 37 degrees Celsius and equilibrate. One milliliter of culture medium for at least one hour. Set up micro drops of each in a Petri dish and overlay with mineral oil.
Now place the culture medium dish into the incubator and the collection medium dish on a slide warmer. Sacrifice the mouse, dissect the ovaries and place them into a watch glass containing prewarm. Collection medium.
Using a one milliliter insulin syringe, anchor the ovaries to the dish and release the antral follicles by puncturing them several times with 27 gauge needles that are fastened together. Under a dissection microscope, collect cumulus cyte complexes using a mouth operated glass pipette. Only collect large antral follicles and not the smaller pre antral follicles or denuded oocytes.
Transfer the complexes to a micro drop of prepared collection medium with a small pipette slightly larger than the diameter of the oocyte. Detach the cumulus cells from the complexes. Transfer the denuded oocytes with a larger pipette into a micro drop of culture medium.
Allow oocytes to recover for at least one hour in the incubator prior to manipulation. Make injection pipettes by pulling bo silicate glass capillary tubing in a mechanical pull. Remove the media chamber from a one well chamber slide.
To create a microinjection platform, place a five microliter drop of collection medium as close as possible to a 0.5 microliter drop of SIR N-A-C-R-N-A or morph fo oligonucleotide injection material cover with mineral oil and place on the microscope stage. Place injection and holding pipettes into the holders and position into the drop of collection medium. Turn on the nitrogen tanks that are connected to the pico injector and anti vibration table.
Open the tip of the injection pipette by gently tapping it against the holding pipette while filling it with medium. Transfer five to 10 oh sites from the incubator to the collection medium drop on the platform. Now set the parameters of the pico injector for an injection volume of five to 10 picoliters while pressing clear.
Move the injection needle to the material drop and fill. Return it to the medium drop. Use the holding pipette to capture an oocyte and align the injection needle oocyte and holding pipette along the x axis.
Under higher magnification, advance the injection pipette through the oocyte, being careful to avoid the nucleus. Once the plasma membrane is pierced, press inject after injection, withdraw the needle, release the oocyte and repeat. Once all the oocytes are injected, return them to the incubator to hold in culture medium for the desired time, which typically ranges from 12 to 24 hours.
Then wash the cytes through several drops of CZB and transfer to a micro drop of CZB under oil and incubate for 16 hours to complete maturation to metaphase of myosis two. Place 750 microliters of CZB containing 100 Micromolar Mastri into the well of an organ culture dish that has water in the outer ring. Now wash the eggs through several drops of CZB plus mastri and then transfer them into the organ culture dish and incubate for an hour.
Fix the eggs in a watch glass containing 2%paraform aldehyde in PBS for 20 minutes at room temperature. Next, transfer eggs into blocking solution and store at four degrees Celsius until processing permeable eggs for 15 minutes at room temperature. Then rinse through several large volumes of blocking solution.
Perform the rest of the procedure on the lid of a 96 well plate that is protected from light in a humidified chamber. At room temperature, transfer eggs to 25 microliter drops of blocking solution for 15 minutes. Then to label centromeres transfer eggs to a drop of blocking solution containing Crest Antiserum at a one to 40 dilution incubate for at least one hour.
Then wash through three drops of blocking solution, incubating 15 minutes in each. Transfer eggs to a drop of blocking solution containing fluoro four conjugated antibody and incubate for one hour. Wash twice and blocking solution to detect chromosomes.
Incubate in a one to 5, 000 dilution of cytx green and blocking solution mount samples in five microliters of vector shield. Put four small spots of petroleum jelly at the intended position of cover slip corners to prevent crushing of the eggs. Place the cover slip and seal with nail polish store in a slide box at four degrees Celsius until processing via confocal microscopy while imaging with a 100 x objective capture 0.4 micrometer steps in the Z plane and image the entire region of the metaphase count centromere using imaging software such as Image J from NIH Euploid Mouse eggs contain 20 kinetic core pairs giving 40 total spots in this z projection of a euploid metaphase two egg DNA is colored in green and K kinetico are colored in red.
The arrows point to two distinct chromat arms indicating overlap at kinetico number 17 and to number 18, Following the microinjection procedure. Other methods like Western blotting real-time PCR spindle configuration and chromosome alignment assessments and in vitro fertilization can be performed in order to answer additional questions like how perturbation of your protein of interests affects miotic maturation and developmental potential. After watching this video, you should have a good understanding of how to manipulate all sites and assess PLO of metaphase two x.
