Time schedule for oocyte production and retransfer
First injection: 200 μl GnRH analog i.p. at 9 a.m. on day −5.
Second injection: 300 μl PMSG i.p. at 1 p.m. on day −3.
Third injection 300 μl hGC i.p. at 2 p.m. on day −1.
The rats are mated overnight on day −1 and the females are checked for a plug at 8 a.m. on the morning of day 0 (Fig. 1).
Oocyte isolation should start at 11 a.m. on day 0 (Fig. 1). Depending on the success of the hormone treatment up to 350 oocytes can be obtained from eight donors.
Strictly keeping to the indicated time schedule is crucial for successful oocyte production. Nonetheless, adjustments might be necessary depending on the dark-light cycle of your animal house (see Note 2 ).
3.4 Induction of Pseudopregnancy
The female recipient rats (foster mothers) are treated with a single dose of GnRH analog at 9 a.m. on day −4 (Fig. 1).
At the evening before oocyte transfer (day 0), the rats are mated with vasectomized males (Fig. 1).
3.5 Isolation of Fertilized Oocytes
Prepare sterile M2 and M16 medium by filtering.
Prepare the cover of a petri dish with droplets of M2 medium, four for each oocyte donor.
Prepare a 6-well plate: 3 ml M2 medium in well #1, a mixture of 1.5 ml M2 medium and 1.5 ml M16 medium in well #3, a mixture of 0.5 ml M2 and 2.5 ml M16 medium in well #4, and 3 ml M16 medium in well #5. Well #2 is reserved for the hyaluronidase treatment and contains a 100 μl aliquot of defrosted hyaluronidase diluted in 2 ml M2 medium (see below).
Prepare a 2 ml syringe with 2 ml M2 medium to be mixed with a vial of hyaluronidase immediately before treatment of the oocytes.
Lay out handling pipette type I and II (see Sect. 2.4.1) and a pipette holder with mouthpiece
Get the dissecting stereomicroscope ready with a fourfold magnification objective.
3.5.2 Isolation of the Oviducts
To isolate the oocytes, the donor females that were mated overnight are sacrificed and the abdominal cavity is opened. The two arms of the uterus are located at both sides.
Locate the oviduct and carefully separate it from both uterus and ovary. Note: It is advisable not to grab the oviduct directly with forceps but rather to hold the ovary. Also avoid pulling the oviduct too strongly because it might cause disruption. If the polyovulation was successful, the ovaries should appear red, not orange or yellow, and the swollen ampulla should be visible.
Place the oviduct in a fresh drop of M2 medium; repeat the aforementioned steps with the second oviduct.
3.5.3 Hyaluronidase Treatment and Oocyte Preparation
After transfer of the oviducts into the prepared medium droplets, the following steps should be conducted under the dissecting stereomicroscope with a 4× magnification objective.
The oocytes should be visible and surrounded by a cumulus mass in a swollen distinct part of the oviduct (ampulla). This part should be isolated using a Dumont forceps and a micro-spring-type scissor. Transfer the swollen part of the oviduct into a fresh droplet of medium and repeat the previous steps with the remaining oviducts.
The swollen parts should be incised making the oocytes to pour out, which then can be collected using type I glass pipettes (see above). All embryos are transferred in 2 ml M2 medium in well #1 of the prepared 6-well plate.
When all oocytes have been collected, they are jointly transferred into the M2 medium containing freshly defrosted hyaluronidase located in well #2 of the 6-well plate. After 1 min of incubation, the follicle cells of the corona radiata should dissociate from the oocytes. Naked cells can now be collected using a type II pipette. This process should be conducted as quickly as possible, since prolonged treatment with hyaluronidase causes the vitelline membrane to get flabby and hard to inject (see Note 4 ).
After hyaluronidase treatment the cells are transferred into well #3 of the 6-well plate containing 50 % M16 medium. This inhibits hyaluronidase activity; therefore it must not be used before this step. In the two subsequent wells of the 6-well plate containing increasing amounts of M16 medium (see above), the cells are cleaned from residual follicle cells by repeated pipetting and transfer steps.
Determine the number of oocytes and transfer them into pure M16 medium in an in vitro fertilization dish. Incubate for 3–4 h at 37 °C.
3.6 Injection of the Oocytes with Lentiviral Particles
Have the vial with the lentivirus ready on dry ice.
Lay out the lentivirus injection needle, the holding pipette, and microloader tips.
Lay out handling pipette type III.
Prepare an in vitro fertilization dish with 1 ml M16 medium, mineral oil, a framed microscope slide, and the microscope with micromanipulators for the holding pipette and the injection needle.
3.6.2 Embryo Injection
The following steps must be conducted under biosafety level 2 conditions.
Prepare a framed microscope slide with a droplet of prewarmed M16 medium under sterile conditions. In this droplet the injection will be conducted.
To avoid contamination, the droplet should be covered with embryo-tested mineral oil. To this end, carefully top the inner lumen of the microscope frame using a 1 ml pipette.
Fill the injection needle using long microloader pipette tips, de-aerate prior to injection.
Install the injection needle in the micromanipulator at the microscope, set the pressure 776 hPa, and ensure permanent flow of virus solution.
Install the holding needle in the other micromanipulator and fill it to two thirds with medium.
Load the embryos into the medium droplet on the microscope slide in batches of 30 using a type III handling pipette.
Pick up single oocytes with the holding pipette and carefully inject every embryo into the zona pellucida, avoiding penetration of the oocyte itself (see Note 5 ).
For retraction of the injection needle it might be helpful to first increase the negative pressure in the holding needle and then retract the needle (see Note 6 ).
Repeat the above steps with the remaining oocytes.
Harvest the oocytes after injection and transfer them into a new in vitro fertilization dish using a type III handling pipette.
Culture the embryos overnight at 37 °C.
3.7 Retransfer of Injected Oocytes into the Oviduct of Foster Mothers
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Narcotics: 10 % Ketamine; 2 % Xylazine.
Sterile surgery conditions and assorted surgical instruments: scissors, forceps, micro-serrefines, Dumont forceps, 2 mm blade spring-type micro-scissors; catgut.
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