The use of antisense oligonucleotides in Xenopus oocytes

Abstract

The ability to manipulate gene expression in Xenopus oocytes and then generate fertilized embryos by transfer into host females has made it possible to rapidly characterize maternal signaling pathways in vertebrate development. Maternal mRNAs in particular can be efficiently depleted using antisense deoxyoligonucleotides (oligos), mediated by endogenous RNase-H activity. Since the microinjection of antisense reagents or mRNAs into eggs after fertilization often fails to affect maternal signaling pathways, mRNA depletion in the Xenopus oocyte is uniquely suited to assessing maternal functions. In this review, we highlight the advantages of using antisense in Xenopus oocytes and describe basic methods for designing and choosing effective oligos. We also summarize the procedures for fertilizing cultured oocytes by host-transfer and interpreting the specificity of antisense effects. Although these methods can be technically demanding, the use of antisense in oocytes can be used to address biological questions that are intractable in other experimental settings.

Fig. 1

Mechanisms of antisense oligo action. (A) Degradation of target mRNA by RNase-H cleavage (phosphodiesters, phosphorothioates, chimeric oligos). (B) Inhibition of translation by steric hinderanee (morpholinos, fully modified oligos).

Fig. 2

Diagram of the host-transfer procedure. Ovary is obtained from females by laparotomy, and full-grown stage VI oocytes are manually defolliculated and cultured in oocyte culture medium. Antisense oligonucleotides are injected at doses of 2–6 ng, depending on the gene of interest. Injected oocytes are cultured 24 hours to allow mRNA and oligo degradation to occur, and are treated with 2µM progesterone. The next day, oocytes are colored with vital dyes and transferred to ovulating host females. Eggs are obtained and fertilized by in vitro fertilization.

Fig. 3

Depletion of β-catenin mRNA in oocytes using a DMED-modified chimera. (A) Phenotype of a stage 28 embryo derived from an uninjected oocyte. (B) Ventralized phenotypes of sibling β-catenin-depleted embryos, injected as oocytes with 400 pg of oligo 303-DMED.