In vitro fertilization of mouse oocytes
this protocol describes the procedure of in vitro fertilisation of mouse oocytes. The procedure is performed at the transgenic core facility by a specialist according to the protocols found here:
This procotol refers to the MRC Harwell guideline procedure for performing in vitro fertilisation of mouse oocytes using fresh or frozen sperm. See attached supplementary documentation.
See attached pdf file for detailed information on media
See attached pdf file for detailed information on equipments needed
1. Sperm is either thawed from a cryopreserved stock or extracted fresh from the cauda epipidymis of the male mouse and incubated for 30 min. at 37 °C in TYH medium.
2. Cumuli containing oocytes are transferred to a 90 µL HTF medium drop on a petri dish, covered by mineral oil (NidOil).
3. 10 µl sperm suspension are added to the HTF drop and the IVF dish is incubated at 37 °C for 4-5 hours. Afterwards, fertilization is assessed by second polar body formation.
4. Sperm thawing and IVF procedures as well as TYH and HTF media preparation are performed according to the protocols described at
5. Following successful IVF, the zygotes are supplemented with fresh KSOM Embryomax media everyday until E5.5
cautionary notes are placed wherever necessary in the protocol.
2 hours from the time of oocyte isolation
Successful fertilization of mouse oocytes resulting in healthy embryos
Takeo T, Nakagata N.Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biol Reprod. 2011 Nov;85(5):1066-72. doi: 10.1095/biolreprod.111.092536.
Javier Martin Gonzalez, Transgenic Core Facility, University of Copenhagen
This is a list of supplementary files associated with this preprint. Click to download.
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Posted 27 Jan, 2020
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