1. The H188A mutation was achieved on the in vitro transcription ready pcDNA-flag-hKDM4A-polyA plasmid using the Quikchange II XL Site-directed mutagenesis kit (Agilent 200521).
2. The mutant plasmid was sequence verified and then both wild-type and mutant mRNA were linearized using SfoI (NEB R0606)
3. this was followed by in vitro transcription, poly(A) tailing and precipitation according to manufacturers instructions of the mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies AM1345).
4. Multiple 10 microliter sized single thaw-and-use aliquots of 1000ng/uL wild-type and H188A mutant Kdm4a polyA-mRNA were prepared and frozen at -80 C.
5. Microinjection of MII oocytes take place 14 hours after hCG injection in M2 medium on a station equipped with FemtoJet® (Eppendorf), CellTram® Air (Eppendorf) and Narishige micromanipulators.
6. Approximately 10 pL of a stock concentration of 1000 ng/uL of either wild-type or H188A Kdm4a poly(A) mRNA was injected into cytoplasm of MII oocytes.
7. Injected oocytes are then transferred to a HTF medium drop and undergo IVF as described in an independent protocol listed here, soon (0.5-1 hr) hour after mRNA injection.
8. The resulting embryos are incubated at 37 °C, 5% CO2 in KSOM-Embryomax medium (Merk Millipore) that is refreshed daily up to E5,5 stage, while observing the progress of development.