The overall goal of this experiment is to improve production efficiency of reconstructed embryos and cloned mice using somatic cell nuclear transfer. This method can help answer key questions in the basic biology, developmental biology, and rescuing endangered animal field. Such as embryonic development, nuclear programming, and genetic research conservation.
The main advantage of this method and technique is that it is possible to generate cloned mice with a practical level of efficiency with simple procedures. After preparing culture medium according to the text protocol, dissolve 1.2 grams of BSA in 10 mL of sterile room temperature water. Add approximately 0.12 grams of mixed ion exchange resin beads to the BSA solution at room temperature with gentle stirring.
When the beads change color from blue green to gold, use a pipette to recover the supernatant solution, then replace the beads with a fresh batch. After confirming that the fresh beads do not change color, recover the solution. Then add 250 microliters of 10.5%sodium bicarbonate.
Using a 0.45 micron filter, sterilize the supernatant, then label it as DBSA stock solution and store it at minus 20 degrees Celsius. After performing euthanasia on female mice subjected to superovulation according to the text protocol, used standard dissection techniques to incise the abdomen to access the reproductive tract. Using small straight scissors and tweezers, extirpate the oviducts and transfer them to a sheet of filter paper to wipe off the blood from the surface.
Next, set the oviducts in mineral oil, then use dissection needles to cut up the ampulla of the oviduct and move the cumulus oocyte complexes coming from the ampulla into 200 microliter drops of HCZB medium with 0.1%hyaluronidase. Incubate the tissue on a warming plate for five minutes. Under a stereomicroscope, confirm that the cumulus cells are released from the cumulus oocyte complexes, then transfer second meiotic or M2 metaphase stage oocytes with some remaining cumulus cells to MKSOM medium containing 0.3%DBSA.
Wash the M2 stage oocytes four times by pipetting up and down in the medium to detach the remaining cumulus cells. Then incubate the oocytes in medium at 37 degrees Celsius and 5%carbon dioxide until enucleation. To isolate the denuded oocytes from the cumulus oocyte complexes, under a stereomicroscope while using a pipette, transfer a small number of the remaining cumulus cells dispersed from the cumulus oocyte complexes to HCZB medium with 6%DBSA.
Place the cumulus cells on the warming plate at 37 degrees Celsius until needed for somatic cell nuclear transfer or SCNT. After preparing PVP medium and CB stock solutions according to the text protocol, use a sterilized pipette with 20 microliters of HCZB medium containing five micrograms per milliliter of CB to wash the M2 stage oocytes. After repeating the wash five times, incubate the oocytes in enucleation solution on the warming plate for 10 minutes.
Next, prepare the enucleation chamber, then use a pipette to transfer the M2 oocytes into the chamber. Add four microliters of enucleation solution and cover the chamber with mineral oil. Under a microscope at room temperature, identify the spindles and chromosomes of the M2 stage oocyte.
Then using a holding pipette and micropipette, orient the oocyte with a pronounced first polar body so that the positioning of the spindle and chromosome is at the 3:00 or 9:00 position. Set the paizo pulse intensity to three to six, and using a micromanipulation pipette, drill to the zona pellucida and open a hole in it. Then with a minimal amount of cytoplasm completely enucleate the spindles and chromosomes.
Under visible light, confirm the absence of chromosomes in the cytoplasm, then with MKSOM medium containing 0.3%DBSA lacking CB, thoroughly wash the enucleated oocytes. Incubate the dish at 37 degrees Celsius and 5%carbon dioxide until cell fusion. To prepare HBJE, add 260 microliters of ice-cold HBJE suspension solution to lyophilized HVJ-E, and pipette up and down until fully suspended.
Prepare five microliter aliquots of the solution and store them at minus 80 degrees Celsius until cell fusion. To carry out cell fusion, use 20 microliters of cell fusion buffer to dilute five microliters of HVJ-E solution. Place the diluted HBJE solution on ice until use.
After preparing the cell fusion chamber as shown here, use a pipette to transfer the enucleated oocytes to HCZB medium in the chamber. Add four microliters of each solution as shown, and use approximately one milliliter of mineral oil to cover the chamber. Place the chamber with the enucleated oocytes at room temperature under a microscope at 400x magnification.
Then using the micromanipulation pipette, transfer the donor cells into the HVJ-E suspension solution. Aspirate the cumulus cells one by one to equally separate them from each other, enabling serial cell fusion. Then with a holding pipette, keep the enucleated oocytes in HCZB medium and use the paizo pulse with the micromanipulation pipette to drill the zona pellucida.
After opening the hole in the zona pellucida, pipette a cumulus cell with approximately five times the volume of the HVJ-E solution and expel it tightly into the oocyte membrane without going through the membrane. Following manipulation of cell fusion, promptly transfer the oocytes to MKSOM medium containing 0.3%DBSA, and move them to an incubator at 37 degrees Celsius and 5%carbon dioxide for one hour. The thereputal steps need to be completed within 10 minutes from placing the enucleated oocytes in HCVB medium under the microscope to returning them to the incubator.
Following cell fusion, under a microscope at 400x magnification check for premature chromosome condensation in the reconstructed oocytes. Transfer the reconstructed oocytes to activation medium containing trichostatin A, or TSA and incubate them at 37 degrees Celsius and 5%carbon dioxide in air for six hours. Observe the formation of pronuclei in the SENT embryos, then apply two more hours of TSA treatment in MKSOM medium with 0.3%TBSA.
Carry out vitamin C treatment according to the text protocol. As seen in this table, the TSA and vitamin C treatment on cloned mice oocytes showed a very high rate of pronuclear formation and developed to the two cell stage under all conditions. Some cloned embryos, which were derived from cumulus cells and developed to the two cell stage were transferred to oviducts of pseudo pregnant females.
This figure shows six cloned offspring that developed out of 72 transferred embryos produced from three pregnant females by the serial treatment of TSA and vitamin C.Finally, as demonstrated here, the treatment with TSA and VC significantly improved the efficiency of in vitro embryonic development to the blastocyst stage. While attempting this procedure it's important to remember that all necessary media and reagents are prepared in advance. The procedure itself a simple to follow and well prepared materials will ensure achieving expected results.
After watching this video, you should have a good understanding of how to produce reconstructed oocytes.
