Abstract
Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.
Highlights
Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals
In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent
Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and stimulated with progesterone to induce maturation
Summary
While the frog is succumbing to the anesthetic, disinfect the surgical area with 70% EtOH and prepare the surgical instruments. The following instruments should be on hand: scalpel handle (#3) and blade (#10 or 11), several pairs of Dumont forceps, Bonn iris scissors (curved or straight), Halsey or Olsen-Hegar micro needle holder, and sutures. We prefer Olsen-Hegar needle holders since the handle contains scissor blades, which can be used to trim sutures without switching instruments. 4. After 10 minutes, check that a surgical plane of anesthesia has been achieved. Anesthetized frogs will stop moving and will not respond to a toe pinch or being turned over. This can include clean gloves (non-latex), lab coats, and surgical masks, depending on personal preference and institutional animal care guidelines
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