Abstract
Visualization of in vivo mRNA localization provides a tool for understanding steps in the mechanism of transport. Here we detail a method of fluorescently labeling mRNA transcripts and microinjecting them into Xenopus laevis oocytes followed with imaging by confocal microscopy. This technique overcomes a significant hurdle of imaging RNA in the frog oocyte while providing a rapid method of visualizing mRNA localization in high resolution.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shav-Tal Y., and Singer R.H. (2005) RNA Localization. J Cell Sci. 118, 4077–4081.
St Johnston D. (2005) Moving messages: the intracellular localization of mRNAs. Nat Rev Mol Cell Biol. 6:363–75.
King, M.L., Messitt, T.J. and Mowry, K.L. (2005). Putting RNAs in the right place at the right time: RNA localization in the frog oocyte. Biol. Cell. 97:19–33.
Forristall C., Pondel M., Chen L., and King M. L. (1995) Patterns of localization and cytoskeletal association of two vegetally localized RNAs, Vg1 and Xcat-2. Development 121:201–208.
Kloc M., and Etkin L.D. (1995) Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. Development 121:287–97.
Cote C.A., Gautreau D., Denegre J.M., Kress T.L., Terry N.A., and Mowry K.L. (1999) A Xenopus protein related to hnRNP I has a role in cytoplasmic RNA localization. Mol. Cell 4:431–7.
Bratu D. P., Cha B. J., Mhlanga M. M., Kramer F. R., and Tyagi S. (2003) Visualizing the distribution and transport of mRNAs in living cells, Proc Natl Acad Sci USA 100, 13308–13313.
Bertrand E. C., Pascal S., Matthias S., Shailesh M., Singer R.H., and Long R.M. (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2:437–445.
Forrest K.M., and Gavis E.R. (2003) Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr Biol. 15:1159–68.
Yisraeli J.K., Sokol S., and Melton D.A. (1990) A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development 108:289–98.
Mowry K.L., and Melton D.A. (1992) Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes. Science 21:991–4.
Deshler J.O., Highett M.I., and Schnapp B.J. (1997) Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum. Science 276:1128–31.
Havin L., Git A., Elisha Z., Oberman F., Yaniv K., Schwartz S.P., Standart N., and Yisraeli J.K. (1998) RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization. Genes Dev. 12:1593–8.
Zhang J., Houston D.W., King M.L., Payne C., Wylie C., and Heasman J. (1998) The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94:515–24.
Houston D.W., and King M.L. (2000) A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus. Development 127:447–56.
Yoon Y.J., and Mowry K.L. (2004) Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin. Development 131:3035–45.
Czaplinski K., Köcher T., Schelder M., Segref A., Wilm M., and Mattaj I.W. (2005) Identification of 40LoVe, a Xenopus hnRNP D family protein involved in localizing a TGF-beta-related mRNA during oogenesis. Dev. Cell 8:505–15.
Messitt T.J., Gagnon J.A., Kreiling J.A., Pratt C.A., Yoon Y.J., and Mowry K.L. (2008) Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes. Dev. Cell 15:426–36.
Wallace R.A., Misulovin Z., and Wiley H.S. (1980) Growth of anuran oocytes in serum-supplemented medium. Reprod Nutr Dev. 20:699–708.
Yisraeli J.K., and Melton D.A. (1988) The material mRNA Vg1 is correctly localized Âfollowing injection into Xenopus oocytes. Nature 336:592–5.
Dumont, J. N. (1972) Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136:153–179.
Acknowledgements
We would like to thank Mowry lab members past and present, who helped to develop these methods. This work was supported by NIH grant # R01GM071049 to KLM.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gagnon, J.A., Mowry, K.L. (2011). Visualization of mRNA Localization in Xenopus Oocytes. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_5
Download citation
DOI: https://doi.org/10.1007/978-1-61779-005-8_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-004-1
Online ISBN: 978-1-61779-005-8
eBook Packages: Springer Protocols