Superovulation protocol

Superovulation is a technique used to get female mice to release more eggs than normal. It is a critical step in collection of morulae and/or blastocysts which can be used as hosts for genetically modified embryonic stem cells (ESCs) during generation of genetically modified mouse models. Morulae are collected at 2.5 days post coitus (dpc) and incubated in vitro overnight for use in injections the next day. Alternatively, blastocysts are collected at 3.5 dpc and used for injections shortly after collection. The injected blasts are implanted in pseudopregnant foster mothers and chimeric pups consisting of genetic contributions from both host blastocyst as well as the embryonic stem cells are born. C57BL/6 mice (B6) are commonly used as blastocyst donors for ESC injection. Below is a protocol developed by Taconic to work well with strains on the Taconic C57BL/6NTac background.

For use with the following Taconic models:

Hormone reconstitution:

  • PMSG is reconstituted from a lyophilized powder at an activity of 2000 IU per vial by adding 2 ml of sterile PBS or sterile 0.9% saline. 50 microliters are then aliquoted into 2 ml conical tubes (totaling approximately 40 tubes per vial). Tubes are labeled and placed in 80 °C freezer.
  • HCG is reconstituted from a lyophilized powder at an activity of 10,000 IU per vial by adding 10 ml of sterile PBS or sterile 0.9% saline. 50 microliters are then aliquoted into 2 ml conical tubes (totaling approximately 200 tubes per vial). Tubes are labeled and placed in -80 °C freezer.

Light cycle:

12hrs light / 12 hrs dark

Animal Specs:

  • Female age: ~4-5 weeks of age (12-16 g) at time of PMSG administration
  • Male age: 6-7 weeks at receipt (they are then practiced with a female for at least 1 week); 8 weeks at initial set-up; tear down at 24 weeks of age. Each male is set up only once per week

Hormone concentrations and injections:

  • Each PMSG tube is diluted with 950 microliters of sterile PBS. Each female is then injected with 0.1 ml. (1 tube each should provide enough hormone for 9 females, ideally 10). Final dose 5 IU/female.
  • Each HCG tube is also diluted with 950 microliters of sterile PBS. Each female is then injected with 0.1 ml. (1 tube each should provide enough hormone for 9 females, ideally 10). Final dose 5 IU/female.
  • All injections are delivered intraperitoneally.

Example schedules:

  • Option 1: PMSG on Day 1 at 1:00 p.m., HCG on Day 3 at 11:00 a.m., Set-up on Day 3 at 2:30 p.m. Collection as desired at day 2.5 or 3.5 dpc.
  • Option 2: PMSG on Day 1 at 3:00 p.m., HCG on Day 3 at 1:00 p.m., Set-up on Day 3 immediately after HCG. Collection as desired at day 2.5 or 3.5 dpc. Example: Collection at 3.5 dpc, starting at 6:00 am. Incubate collected embryos for maximum of 1 hour at room temp. For example, for desired Sunday plug: PMSG on Thursday, HCG on Saturday, Set-up on Saturday.
  • Add 1 day to all corresponding parameters for each consecutive plug date.

Notes for blastocyst injections:

  • Taconic has found injection of 7-9 B6-derived ES cells per blastocyst to be optimal.
  • It is important to inject embryos in the best phase (not under/over- developed). Chimerism is strongly dependent on this factor.