Culture of Experimental Oocytes

Prepare ovarian tissue surgically from anesthetized females as described in Schneider et al. (2010) and Olson et al. (2012) and place in OCM at 18°C.

Alternatively, Xenopus ovarian tissue can be obtained commercially.

Manually defolliculate 300–500+ oocytes using Dumont forceps. Dissect oocytes by tearing the follicle layer near the region where oocytes are attached to the ovary, using very light grip pressure on the forceps. Store oocytes in OCM at 18°C and begin experiments within 1–2 d.

Frogs stimulated with hGC within 3 mo should not be used as oocyte donors. Healthy, high-quality oocytes are essential for successful host-transfer experiments. These should be fully grown and free of any damage to the membrane. Practice is essential for the rapid collection of many good quality oocytes and is necessary for good results with this method. For detailed descriptions of manual defolliculation as well as videos see: Smith et al. (1991), www.youtube.com/watch?v=us8rDNG69Sk(Manual defolliculation 2009), Protocol: Isolation of Xenopus Oocytes (Sive et al. 2010a), Hulstrand et al. (2010), Schneider et al. (2010), Olson et al. (2012), and Protocol: Isolation of Xenopus Oocytes (Newman et al. 2018).

Microinject oocytes directly while in OCM; Ficoll is not needed. Transfer the oocytes to fresh OCM after injection and culture for up to 3 d at 18°C. Include appropriate controls, such as uninjected oocytes and mRNA rescued oocytes, in the case of oligonucleotide depletions, or control mRNAs for overexpression experiments.

For a detailed description of microinjection see: Protocol: Microinjection of Xenopus Oocytes (Sive et al. 2010b). Antisense oligonucleotides are typically injected 36–48 h before fertilization, whereas mRNAs can be injected the day before host transfer and fertilization. Typical doses for antisense oligonucleotides are 2–6 ng for modified DNA-based oligonucleotides and 10–50+ ng for morpholino oligonucleotides. The optimal dose should be empirically determined in pilot studies. For overexpression/gain-of-function studies, high doses of mRNAs of 1 ng or more can be used. When rescuing a mRNA knockdown effect, minimal doses are used (20–200 pg), ideally below the phenotypic threshold, so that rescue of the depletion can be accurately assessed as opposed to an ectopic overexpression effect. For genome editing, equimolar amounts ( 2 µM each) of Cas9 protein and appropriate guide RNAs are assembled for 10 min at 37°C and 300 pg sgRNA/1.5 ng Cas9 protein are injected.