Preparation of a highly active cell-free translation system from immature Xenopus laevis oocytes
Introduction
The study of eukaryotic translation has been greatly aided by the generation of cell-free systems that are based upon a variety of organisms and tissues [1], [2], [3]. In addition, in vitro translation systems have been reconstituted from purified components of both Saccharomyces cerevisiae and rabbit reticulocytes [4], [5]. These systems offer unique advantages to the study of translation: while in vitro systems reconstituted from purified factors have been extensively used to detail the mechanisms underlying general translation [6], [7], cell-free systems have been extremely useful in analyzing cell-specific gene expression at the level of translation [2], [8]. For example, regulatory components present in germ cells are likely absent or modified in somatic tissues, and vice versa.
Cell-free systems from eggs and fully-grown oocytes (stage VI; [9]) of the frog Xenopus laevis have been widely used to study a variety of cellular processes including translation, translocation of secretory proteins, nuclear envelope assembly, DNA replication and repair, and cell cycle [10], [11], [12], [13]. The wide use of Xenopus oocytes and eggs for the preparation of these systems stems in part from the ease of acquisition of large amounts of material from these animals. A single female frog can yield thousands of oocytes that, due to their large size (50–1300 μm in diameter), can be easily sorted by stages (I–VI; [9]).
During oogenesis, numerous maternal mRNAs are stored within the oocyte in preparation for fertilization and early embryogenesis when the zygotic genome is transcriptionally inactive [14], [15]. The production of protein from these mRNAs is stringently regulated, since all cellular processes in the early stages of embryogenesis are reliant on maternally loaded transcripts and proteins. In view of the fact that these mRNAs are stored in the oocyte but not translated until after maturation of the fully-grown oocyte or upon fertilization of the egg, they must be maintained in a translationally silent, or “masked” state. A number of studies have elucidated the mechanisms underlying the translational regulation of key mRNAs in the late stages of oogenesis [16], [17], and this work has been much assisted by the in vitro translation systems developed for eggs and fully-developed Xenopus oocytes [13].
A number of mRNAs in Xenopus oocytes are localized early in oogenesis (stages I–III; [18], [19]). mRNA localization results in a local enrichment of a particular mRNA, and ultimately of the encoded protein, and this spatial restriction of translation is crucial for the development of the embryo [20]. It is therefore critical that no protein be produced from these mRNAs until they have reached their cellular destination. The mechanisms controlling translation of localized mRNAs in early oogenesis remain largely unknown, although a number of cis- and trans-acting factors have been identified through in vivo injection approaches [16], [21]. In order to conduct mechanistic analyses of translational control mechanisms acting during early oogenesis, we have developed a cell-free translation system from stage I to stage III Xenopus oocytes. The lysate produced using this protocol is highly active, and can translate an exogenously supplied reporter mRNA with an efficiency equal to or greater than the commercially available rabbit reticulocyte lysate system. Given the high activity of this oocyte lysate and the ready availability of large amounts of Xenopus oocytes, this cell-free translation system represents a useful tool both for general studies of translational regulation and for understanding mechanisms of translational control in oocytes.
Section snippets
Isolation of oocytes
For the preparation of an active translation lysate from stage I to stage III oocytes, it is necessary to isolate a large number of oocytes from the early stages of oogenesis. First, the entire ovary of a young X. laevis female (6.25–7.5 cm juveniles; Nasco LM00714MX) is surgically removed [22] and placed in MBSH buffer [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4·7H2O, 0.33 mM Ca(NO3)2·4H2O, 0.41 mM CaCl·6H2O, 10 mM HEPES (pH 7.6)]. The ovaries of frogs of this age contain predominately oocytes
Concluding remarks
We have described the preparation of XTL, a cell-free translation system using oocytes from the early stages of oogenesis in the frog, X. laevis (Fig. 1). The XTL system can be used to assay translation from an exogenously added reporter mRNA (Fig. 2, Fig. 3), and to analyze regulatory elements in the UTRs of similar reporters. The translation efficiency of XTL is extremely high, and is comparable with commercially available in vitro translation systems (Fig. 2). Previously developed systems
Acknowledgment
Work in our laboratory on development of these methods was supported by NIH Grant # R01GM071049 to K.L.M.
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Cell-free protein synthetic system: progress and applications in biopharmaceutical engineering
2014, Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology