The University of Iowa, Department of Biology, 257 BB, Iowa City, IA 52242-1324, USA.
Methods. 2010 May;51(1):75-81. doi: 10.1016/j.ymeth.2009.12.015. Epub 2010 Jan 5.
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.
利用 Xenopus 卵母细胞中基因表达的调控,然后通过将其转移到宿主雌体中产生受精卵,这使得快速鉴定脊椎动物发育过程中的母体信号通路成为可能。特别是通过内源性 RNase-H 活性介导的反义脱氧寡核苷酸(oligos),可以有效地耗尽母体 mRNA。由于在受精后将反义试剂或 mRNA 微注射到卵子中通常不能影响母体信号通路,因此 Xenopus 卵母细胞中的 mRNA 耗尽非常适合评估母体功能。在这篇综述中,我们强调了在 Xenopus 卵母细胞中使用反义的优势,并描述了设计和选择有效 oligos 的基本方法。我们还总结了通过宿主转移受精培养卵母细胞的程序,并解释了反义效应的特异性。尽管这些方法在技术上可能具有挑战性,但在卵母细胞中使用反义可以用于解决在其他实验环境中难以解决的生物学问题。