This protocol falsely dates the modification of genomic imprinting in Haploid mouse embryonic stem cells, as our subject's due to false sperm, enabling the generation of home-grown embryos in mice. The genetic modification of a gamete generally require difficult techniques. Using this methodology, parental gametes can be manipulated in a hybrid embryonic stem cells for subsequent introduction into mice as a sperm replacement option.
As this method enables our artificial specific modification of a paternal gamete in embryos, it is particularly applicable to study of genomic imprinting and cauterization. Demonstrating this procedure will be Charles Etienne Dumeau, a mouse embryologist from my laboratory. To prepare holding and microinjection pipettes for the experiment, first, use a micropipette puller to pull borosilicate glass capillaries with an elongated shape and a gradual taper.
To prepare holding pipettes, position a pulled capillary with an outer diameter of 60 to 100 micrometers in a micro forge over the glass bead on the filament. Switch on the filament and lower the capillary until it contacts the bead. When the capillary has attached to the bead, switch off the filament and retract the capillary, causing the capillary to break with a straight end.
Next, position the broken capillary tip horizontally next to the glass bead on the filament and heat the filament to melt the end until the inner diameter of the capillary tip reaches 10 to 20 micrometers in diameter. Then position the capillary above the glass bead approximately one millimeter from the tip and heat the filament to allow the capillary to be bent to a 20 degree angle. To prepare a micro injection pipette, position a pulled capillary with an outer diameter of about six micrometers above the glass bead on the heated filament and lower the capillary until it contacts the bead.
Once the capillary has attached, switch off the filament and retract the capillary. The capillary will break with a straight end. Then move the capillary over the glass bead approximately one millimeter from the capillary tip and heat the filament to allow the capillary to be bent to a 20 degree angle.
To perform an intracytoplasmic injection, place five microliter drops of PVP solution and 20 microliter drops of M2 medium on the upside down lid of a 10 centimeter dish and cover the drops with mineral oil. Place the dish onto the stage and install a holding pipette onto the micromanipulator. Use a micro loader tip to fill the microinjection pipette with fluorocarbon oil and mount the pipette onto the piezo actuator.
Observing through the microscope, lower the microinjection pipette into a drop of PVP solution. When the pipette is immersed, pipette up and down several times to coat the glass with the PVP. Load a small volume of PVP solution into the microinjection pipette.
Immerse the microinjection and holding pipettes in the M2 medium and focus on the pipette at the bottom of the drop. Transfer approximately two microliters of the double knockout parthogenic ES cell suspension into the M2 medium drop and use a mouth pipette to transfer 10 M2 oocytes into the same drop. Rotate one oocytes and apply negative pressure through the holding pipette to hold the oocyte so that the perivitelline space faces the microinjection pipette, taking care that the metaphase two plate is not positioned in the path of the microinjection pipette.
Draw one double knockout parthogenic ES cell into the tip of the microinjection pipette and confirm the rupture of the cell membrane. Apply light negative pressure to the microinjection pipette and deliver four piezo impulses while pushing the tip of the microinjection pipette toward the perivitelline space to break through the zona pellucida. Penetrate the oocyte with a microinjection pipette so that the oolemma stretches to the opposite side and apply one piezo pulse to pierce the oolemma, making sure that the oolemma it relaxes along the shaft of the microinjection pipette.
Inject the double knockout parthogenic ES cell with a minimal volume of medium into the ooplasm and withdraw the microinjection pipette smoothly from the oocyte. Release the injected oocyte from the holding pipette and move it to the side of the micro drop for later collection. When all of the oocytes have been injected, transfer the oocytes to a prewarmed center well dish containing KSOM medium and incubate the dish at 37 degrees Celsius and 5%carbon dioxide for one hour before activation.
DNA content analysis of this representative double knockout parthogenic ES cell line, carrying the CAG/EGFP transgene by flow cytometry, illustrates the distribution of haploid and diploid cells at the G0, G1, S and G2 M phases. Genotyping can be performed to confirm the absence of wild type alleles at the H19 and intergenic differentially methylated regions. A CAG/EGFP transgene can be introduced into double knockout pathogenic ES cells to study their contribution to semi-cloned embryos by the visualization of green fluorescence under a microscope.
Flow cytometry analysis shows two populations corresponding to the G2 M phase arrested haploid and diploid cells after demuculcene treatment. The absence of a 1N haploid peak indicates that the cell's cycle of rest was largely complete. After injection, EGFP expression is rarely detected in the constructed semi-cloned embryos.
as the cytoplasm of double knockout parthogenic ES cells disperses into the large cytoplasm of the oocyte. Six hours after activation up to three polar bodies can be observed under the microscope. The first and second polar bodies of the oocyte and one pseudo polar body from the haploid ESC.
To demonstrate their developmental competence, semi-cloned embryos can be cultured to the blastocyte stage. Furthermore, full-term female mice can be obtained from semi-cloned two cell stage embryos transferred into the oviducts of recipient females. During micro injection it is important to rupture the plasma membrane of haploid embryonic stem cells as it figures in to prevent chromosome segregation when the oocyte is activated.
Instead of parthenogenetic haploid ES cells, androgenic haploid ES cells can also be used. allowing either the maternal or paternal gamete to be selected as the sperm replacement depending on the study purpose.
