Somatic Cell Cloning Efforts in the Monkey
As noted previously, nuclear reprogramming is thought to be the limiting parameter in our monkey somatic cell cloning protocol. Obviously, the reprogramming required after SCNT is more extensive than the process that occurs during embryonic cell NT. For normal full term development of cloned embryos, genes normally expressed during embryogenesis, but silent in the somatic donor nucleus, must be reactivated in an appropriate temporal and spatial manner. Despite the fact that even terminally...
Fertilization by ICSI and Embryo Culture
ICSI is a robust efficient fertilization procedure in the monkey resulting in high pronuclear formation rates (80-90 ). 1. Wash collected spermatozoa twice by resuspending with TH3 medium followed by centrifugation of the liquid portion of the ejaculate for 7 min at 200g. 2. Take an aliquot and determine motility and concentration before the final cen-trifugation and resuspension step. 3. Adjust sperm concentration to 1 x 106 motile spermatozoa per milliliter in TH3 medium and store for approx...
Medicen Colozen
Means with a different superscript are significantly different (p > 0.05). MOET, multiple ovulation embryo transfer AI NM, artificial insemination natural mating. Means with a different superscript are significantly different (p > 0.05). MOET, multiple ovulation embryo transfer AI NM, artificial insemination natural mating. 1. Recipient pregnancy monitoring there needs to be some forethought about recipient selection. Multiparous cows are superior to heifers in their ability to calve per...
Paul J Verma Alan O Trounson
Production of Cloned Calves Using Roscovitine-Treated Adult Somatic Cells as Donors Kazuchika Miyoshi, Sezen Arat, and Steven L. Stice The stage of the donor cell cycle is a major factor in the success of cloning. Quiescent cells arrested in the Gq Gj phases of the cell cycle by either serum starvation or growth arrest when cultured cells reach confluence have been used as donors to produce cloned animals. Recently, we have developed a novel and effective method using roscovitine to synchronize...
Preparation of Cytoplasts and Somatic Cell Suspension
Removal of the Cumulus Cells and the Zona Pellucida 1. Remove 150-200 oocytes from the maturation dish 21 h after the start of in vitro maturation (hpm) and transfer to an Eppendorf tube containing 500 L of hyaluronidase solution. 2. Mix suspension carefully with a 1000 L of automatic pipet until all visible cumulus clouds disappear. 3. Vortex the tube at maximum speed (8000 rpm) for 3 min. 4. Collect oocytes in a 36-mm diameter Petri dish under a stereo microscope and wash twice in T2...
