Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technology for the treatment of sperm-related infertility complications. ICSI is used to enhance the fertilization phase of in vitro fertilization (IVF) by injecting a single sperm into an MII oocyte. ICSI only requires one sperm cell per oocyte as the acrosome reaction is skipped with this procedure, while for IVF thousands of sperm are needed that compete with each other to fertilize the oocyte.
The TMII core will separate the sperm head from its tail and prepare it for injection. MII oocytes are then injected with sperm, incubated for 5-7 hours after which the presence of or absence of pronuclei in the oocytes will be determined. Once the presence of the pronuclei is confirmed, the oocytes will be cultured until they reach the 2-cell stage (24–30 h after microinjection). ICSI oocytes will then be transferred into the oviducts of 8-week-old surrogate pseudopregnant CD-1 recipient females at 0.5 days post coitus (dpc). Pregnant females are allowed to deliver and raise their pups. The pups will then be transferred to the investigator.
Please note that there is no guarantee of live pups being produced. We will do our utmost to produce live pups, but we have no way of controlling the health of the donor or how the sperm samples were frozen, stored, handled, or shipped before we received them, therefore we cannot guarantee that the frozen sperm will be viable for rederivation.
Somatic cell nuclear transfer (SCNT) is a technique for cloning. The nucleus is removed from a healthy oocyte. This oocyte becomes the host for a nucleus that is transplanted from another cell, such as a tail fibroblasts or other somatic cells. The resulting embryo can be used to generate embryonic stem cells with a genetic match to the nucleus donor (therapeutic cloning), or can be implanted into a surrogate mother to create a cloned individual, such as Cumulina, the first mouse ever cloned in 1998 using the Honolulu method (reproductive cloning).
Please contact the TMII. See more Services.
In vitro fertilization is the most commonly used technique in order to recover a mouse line from frozen sperm. It can also be performed with fresh sperm in order to generate a high number of 2-cell embryos which can be either cryo-preserved or transferred to a surrogate pseudopregnant CD-1 recipient females. It can help to speed up the re-derivation process of a mouse line or to quickly expand a mouse colony, generating a big number of pups born at the same time. For male mice with lower sperm activity (ex. Aging), a sample of fresh sperm can be taken from the male, providing an alternative to natural mating without having to sacrifice the mouse.
For best results at reanimation of frozen sperm, we strongly recommend that clients provide at least two straws. Frozen sperm must be shipped in a liquid nitrogen dry shipper and stored in liquid nitrogen (not vapor phase) upon arrival. Please note that there is no guarantee of live pups being produced. We will do our utmost to produce live pups, but we have no way of controlling the health of the donor or how the sperm samples were frozen, stored, handled, or shipped before we received them, therefore we cannot guarantee that the frozen sperm will be viable for rederivation.
Females are superovulated by PMSG/ HCG and their eggs are collected 13 hours post HCG. Capacitated fresh or thawed sperm is used for fertilizing eggs. After 4 hours of incubation at 37oC, eggs are washed and incubated in KSOM overnight at 37oC. 2-cell embryos are then transferred to surrogate pseudopregnant CD-1 recipient females. All weaned pups will be transferred to the investigator. The investigator is responsible for the genotyping of the pups and determination of which pups have the desired genotype(s). Fertilization rate varies with strains, it can be up to 90% when using sperm and oocytes from an F1 hybrid strain i.e. CBA x C57BL6. However, it may drop to 5% in inbred strain such as C57BL6 and 129. Thus, we cannot offer a guarantee that any given IVF procedure will produce large number of pups. The standard recharge for each IVF session is the cost of purchasing the egg donors and a fee to recover our costs. If both the experimental and the control IVF procedures fail then we will not recharge your account.
Embryonic stem (ES) cells are injection of into tetraploid mouse embryos to avoid the poor efficiency of the nuclear transfer, a key limitation of previously established cloning techniques. Specifically, the cells of an embryo in the two-cell stage are joined by cell fusion into a single cell with twice the chromosome count. As a result, these cells are no longer in a position to form a complete living organism but are still able to differentiate into trophectoderm, that is to the enveloping layer of the embryo in the blastocyst stage, which later forms the placenta and the umbilical cord. To form a living organism, though, it also needs the inner cell mass of the blastocysts, the embryoblast. Once the trophoblast cells have been supplemented by embryonic or induced pluripotent stem cells, these in turn build the inner cell mass of the blastocysts, i.e. the embryoblast. These complete embryos are then transferred to the surrogate pseudopregnant CD-1 recipient females and some of them grow into viable mice after birth. At day 20, the fetus will be delivered by cesarian section and placed with a foster mother until weaning.
Please contact the TMII. See more Services.