Microinjection into Xenopus oocytes

Mertz, J. E., and Gurdon, J. B. (1977). Purified DNAs are transcribed after microinjection into Xenopus oocytes. Proc. Natl Acad. Scl U.S.A. 74, 1.502-1506. 

As with other in vitro systems, different mRNAs exhibit differing translational efficiencies in the Xenopus extract. Where comparisons have been possible, relative translational yields in the extract reflect those seen on microinjection into Xenopus oocytes. Natural mRNAs normally translate best, while the performance of most synthetic mRNAs is much enhanced when transcribed from the vector pSP64T (Krieg and Melton, 1984), where the cDNA is flanked by sequences from the Xenopus P-globin cDNA. A reliable protocol for the synthesis of capped transcripts in vitro is given in Matthews and Colman (1991). 

On the other hand, Asselbergs et al. (1980) observed translational competition between globin mRNA, lens crystallin mRNAs, and turnip yellow mosaic virus RNA in the micrococcal nuclease-treated reticulocyte lysate, but not after microinjection into Xenopus oocytes (Asselbergs et al., 1979). The reason for this discrepancy is not clear, as Laskey et al. (1977) have shown that in oocytes, a component of the translation machinery, and not mRNA, limits the rate of protein synthesis. 

Le Gaherec F, Bron P, Verbavatz JM, Garret A, Morel G, et al. 1996. Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection functional characterization of a novel aquaporin. J Gell Sci 109 (Pt 6) 1285. 

There is recent data to suggest that there may in fact be a biological role for the internalized insulin and EGF receptors (both of which are themselves tyrosine kinases). Thus, microinjection of insulin-occupied insulin receptors into Xenopus oocytes causes the increased phosphorylation of ribosomal protein S6 (a known substrate for the insulin receptor/kinase) [62] and the EGF receptor in endocytic vesicles has been shown to retain its kinase activity [63]. Whether the internalized insulin receptor/kinase or EGF receptor/kinase has a physiological role or not is as yet unknown. Clearly, though, these data suggest that there is much more to be learned about the role of internalized hormone-receptor complexes, especially those where the receptor possesses intrinsic enzymatic activity. 

Currently, it is standard procedure to develop ion channel-specific antibodies for immunocytochemistry, to perform Western and Northern blot analyses, ion channel in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR). The introduction of the single-cell RT-PCR in combination with the patch-clamp method in the 1990s made it possible to identify gene transcripts and to correlate them with functional data for the same individual cell. Finally, one of the most powerful cell biological techniques in the study of ion channels is based on artificial expression systems such as microinjection of mRNA encoding channel subunits into Xenopus oocytes and selective expression of native ion channels or with different subunit composition (e.g., Ky channel subunits). Because the Xenopus oocytes are large, they are a perfect model to study artificially expressed channels. Another good model for artificial ion channel expression is the Chinese hamster ovary (CHO) cell line. 

Import of aminoacyl-tRNA into living cells is another approach toward in vivo production of nonnatural mutant proteins. Dougherty and coworkers microinjected [41] or electroporated [44] an aminoacyl-tRNA/mRNA pair into Xenopus oocyte to synthesize fluorescently labeled acetylcholine receptor. The microinjection method is applicable to any type of tRNA and amino acid, but the number of cells that can be treated at one time is very limited. 

This article has described methods in use in our lab for microinjection of genes into Xenopus oocyte nuclei followed by EM visualization of those genes by the... 

Simons, F. H., Pruijn, G. J and van Venrooij, W. J. (1994). Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes. J. Cell Biol. 125, 981-988. 

Stefanovic, B., Hackl, W., Luhrmann, R., and Schumperli, D. (1995). Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes. Nucleic Acids res. 23, 3141-3151. 

Spivack JC, Erikson RL, Mailer JL. 1984. Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation. Mol Cell Biol 4(8) 1631-1634. 

Grosjean H, Kubli E (1986) Functional aspects of tRNAs microinjected into Xenopus laevis oocytes results and perspectives. In Celis JE, Graessmann A, Loyter A (eds) Microinjection and organelle transplantation techniques. Academic, London, p 301... 

A EXPERIMENTAL FIGURE 21-6 A diffusible factor in arrested Xenopus eggs promotes meiotic maturation. When 5 percent of the cytoplasm from an unfertilized Xenopus egg arrested in metaphase of meiosis II is microinjected into a G2-arrested oocyte (step lEI), the oocyte enters meiosis I (step B) and proceeds to metaphase of meiosis II (step B), generating a mature egg in the absence of progesterone. This process can be... 

Fig. 6 (A) The export of tRNA and ribosomal subunits is saturable. The results are representative of three experiments in which at least 10 oocytes were analyzed for each time point. Error bars indicate standard deviation and are sometimes too small to visualize on these graphs. (A) In vitro transcribed P-labeled RNA (6 nM was injected into the nuclei of Xenopus oocytes, and the time course of export was assayed in the absence ( ) or presence ( ) of 60 p,M unlabeled tRNA. (B) IRNA export is stimulated by coinjection with 40S ribosomal subunits or purified rRNA. P-Labeled tRNA (6 nAf) was microinjected into nuclei in the absence ( ) or presence ( ) of purified ribosomal RNA (1.6 p-M). [Reprinted with permission from Pokrywka and Goldfarb (1995).)... 

For in vitro reconstitution experiments labeled or nonlabeled lamin A (0.6 ml at 0.1-0.15 mg/ml 8.0 M urea, 20 mM sodium phosphate, pH 8.0) was dialyzed overnight at 4°C against lamin A reconstitution buffer [20 mM 2-(/V-morpho-lino)ethanesulfonic acid, 175 mM NaCl, 1 mM DTT, 0.2 mM PMSF, pH 6.5]. The reconstituted polymers were pelleted for 15 min at 13,000 g. Pellets were fixed and processed for electron microscopy (see Section II,A,3). Lamin LI does not form paracrystals under the reconstitution conditions described above. Therefore the assembly properties of lamin LI were tested by microinjection of the dialyzed 5-IAF-labeled protein into the cytoplasm of Xenopus oocytes, fol-... 

Microinjection of Cdc25 protein phosphatase into Xenopus prophase oocyte activates MPF and arrests meiosis at metaphase I. Biol Cell 82(l) ll-22. 

---