Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes.

Localization of a specific subset of maternal mRNAs to the vegetal cortex of Xenopus oocytes is important for the regulation of germ layer formation and germ cell development. It is driven by vegetal localization complexes that are formed with the corresponding signal sequences in the untranslated regions of the mRNAs and with a number of different so-called localization proteins. In the context of the present study, we incorporated tagged variants of the known localization protein Vg1RBP into vegetal localization complexes by means of oocyte microinjection. Immunoprecipitation of the corresponding RNPs allowed for the identification of novel Vg1RBP-associated proteins, such as the embryonic poly(A) binding protein, the Y-box RNA-packaging protein 2B and the oocyte-specific version of the elongation factor 1α (42Sp50). Incorporation of 42Sp50 into localization RNPs could be confirmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50. Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNAse-sensitive complexes, as well as to associate specifically with several vegetally localizing mRNAs but not with nonlocalized control RNAs. Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between the nucleus and cytoplasm. Taken together, these observations provide evidence for a novel function of 42Sp50 in the context of vegetal mRNA transport in Xenopus oocytes.

Elr-type proteins protect Xenopus Dead end mRNA from miR-18-mediated clearance in the soma.

Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.

Generation of transgenic frogs.

The possibility of generating transgenic animals is of obvious advantage for the analysis of gene function in development and disease. One of the established vertebrate model systems in developmental biology is the amphibian Xenopus laevis. Different techniques have been successfully applied to create Xenopus transgenics; in this chapter, the so-called meganuclease method is described. This technique is not only technically simple, but also comparably efficient and applicable to both Xenopus laevis and Xenopus tropicalis. The commercially available endonuclease I-SceI (meganuclease) mediates the integration of foreign DNA into the frog genome after coinjection into fertilized eggs. Tissue-specific gene expression, as well as germline transmission, has been observed.

Transgenesis procedures in Xenopus.

Stable integration of foreign DNA into the frog genome has been the purpose of several studies aimed at generating transgenic animals or producing mutations of endogenous genes. Inserting DNA into a host genome can be achieved in a number of ways. In Xenopus, different strategies have been developed which exhibit specific molecular and technical features. Although several of these technologies were also applied in various model organizms, the attributes of each method have rarely been experimentally compared. Investigators are thus confronted with a difficult choice to discriminate which method would be best suited for their applications. To gain better understanding, a transgenesis workshop was organized by the X-omics consortium. Three procedures were assessed side-by-side, and the results obtained are used to illustrate this review. In addition, a number of reagents and tools have been set up for the purpose of gene expression and functional gene analyses. This not only improves the status of Xenopus as a powerful model for developmental studies, but also renders it suitable for sophisticated genetic approaches. Twenty years after the first reported transgenic Xenopus, we review the state of the art of transgenic research, focusing on the new perspectives in performing genetic studies in this species.

I-SceI meganuclease-mediated transgenesis in Xenopus.

Several experimental approaches have been described to generate transgenic frogs. Here, we report on the application of a novel method in Xenopus, making use of I-SceI meganuclease. The characteristic feature of this endonuclease is that it has an extended recognition site of 18 bp, which is expected to exist only once in 7 x 10(10) bp of random DNA sequences. Various reporter constructs flanked by two I-SceI recognition sites were injected together with the I-SceI meganuclease into one-cell stage Xenopus embryos. We observed an overall transgenesis frequency of 10% or more under optimized condition. The injected genes were integrated into the genome and transmitted to F1 offspring. Southern blot analysis showed that between one and eight copies of the transgene were integrated. Meganuclease-aided transgenesis, thus, provides a simple and highly efficient tool for transgenesis in Xenopus.