Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

The overall goal of this procedure is to assess myotic spindle formation and organization in Cytes following irna mediated depletion of key MTOC associated proteins such as perrin. This method can help answer key questions in a reproductive biology field, such as the mechanisms regulating chromosome segregation and potential disruptions that lead to aneuploidy in all sites. The main advantage of this technique is that it results in efficient transcript, knockdown, and specific immunofluorescence analysis of individual cytes.

This procedure can also be adapted to study almost any target protein 48 hours before the collection day treat The female mice used for oocyte collection with five international units of PMSG injected intraperitoneal after collecting the ovaries using standard practices. Transfer to a dish of prewarm equilibrated, MEM media supplemented with BSA and mil reone and prepare to isolate the cytes under a dissecting microscope. Release the cumulus cyte complexes or COCs by fixing an ovary to the bottom of the culture dish using a 27 gauge needle and use a second needle to puncture the antral follicles using oocyte aspiration or standard mouth pipetting practice.

Collect the oocytes surrounded by two or more layers of compact cumulus cells. Transfer these COCs to a new dish with M-E-M-B-S-A mild reone and incubate them for an hour at 37 degrees Celsius. Then proceed with denuding the cytes using standard methods.

Prepare the injection culture dish with three milliliters of M two media with one microgram per milliliter of milone. Also prepare the washing and culturing dishes with M-E-M-B-S-A milone. Next load five microliters of the one millimolar INA solution into the injection needle and set it up for the micro injection.

Now place a 100 microliter drop of M two media on the lid of a three centimeter dish. Load one oocyte into the drop and use it to adjust the position of the holding pipette. Secure the OAS site by attaching it to the holding pipette using negative pressure.

Then adjust the position of the injection needle to the widest diameter on the oocyte. Once positioned, inject about 10 picoliters of solution into the oocyte cytoplasm, avoiding the nucleus. Return the lid to the stereo microscope stage and add 100 microliters of fresh M two media.

Put about 10 cytes into the media. Then on the micro injection stage, proceed with the micro injection. Secure each oocyte with the holding pipette and slowly insert the injection needle into the cytoplasm.

Inject the S irna solution and carefully retract the injection pipette. Move successfully injected cytes to one end of the drop and move unsuccessfully injected cytes to the opposite end of the drop. It should only take a few minutes to inject 10 cytes, which is critical to maintain their viability.

Move the lid to the stereo microscope stage and transfer all the successfully injected cells to a dish of M-E-M-B-S-A Mel known at 37 degrees Celsius. Transfer cytes to a 37 degree Celsius incubator with a medical gas atmosphere and incubate for 24 hours. Begin with releasing the cytes from the mil reone block by washing the cells three times in M-E-M-B-S-A.

Then transfer the cytes to maturation medium containing 10%FBS culture. The cells for 17 hours at 37 degrees Celsius. So meiosis will resume the next day.

Fix the cytes. Use cyte aspiration or mouth pipetting to swiftly move the different experimental groups to wells of four well plates containing 750 microliters of prewarm 4%PFA. Let the cytes incubate for an hour later.

Wash each group three times with 750 microliters of warmed PBS with 5%FBS. Each wash should go on for 15 minutes in the incubator after washing block each O site group in 5%FBS in BS.Let the block go on overnight at four degrees Celsius to immunostain. Use a row of eight wells on the multi-well plate for each experimental group of cytes.

4 96 well plates load 200 microliters of solution per well. Begin the staining process with freshly prepared primary antibody solution. Then cover the wells with param and incubate the cytes according to the antibody specification following the primary antibody.

Wash the cytes three times with 5%FBS in PBS for 10 to 15 minutes per wash. After three washes, transfer the cytes to a well of secondary antibody solution. Cover these wells with paraform.

Incubate the cells for an hour at 37 degrees Celsius and then wash three times as before. Once washed, transfer the oocytes to clean glass slides. All the oocytes in one experiment group are transferred to a single slide.

Aspirate any excess solution to immobilize the cells and immediately add eight microliters of dappy containing mounting media. Then cover, slip them carefully to avoid air bubbles or damage to the cytes cytes were micro injected with a nonspecific control or perrin irna after 17 hours of culture, most controls reached the metaphase two stage and contained organized myotic spindles with aligned chromosomes. Perrin labeling shown in red was bright and focused at the spindle poles.

Transcripts were successfully knocked down as perrin staining in the irna injected cells was not detected. Most of these oocytes remained in metaphase one and exhibited disorganized spindle structures with misaligned chromosomes. A few progressed to metaphase two, but also exhibited disrupted spindles.

Now you should have a good understanding of how to collect and micro inject mouse cytes with specific as irna and then immuno stain these cytes for fluorescence analysis of spin formation and organization. It's important to remember to work swiftly and with great care to preserve oside viability and Quality.