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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.
RNA localization is a conserved mechanism of establishing cell polarity in a variety of cell types and organisms. Such spatial regulation of gene expression can define specialized regions of the cell, and prominent examples include germ layer specification during vertebrate development and cytoskeletal rearrangements involved in cell motility (1–3).
Visualization of RNA distribution patterns has provided valuable insights into RNA transport steps and mechanisms. A number of techniques have been developed to visualize subcellular RNA localization, including in situ hybridization with digoxigenin- and fluorescently-labeled probes (4–6), molecular beacons (7), and fluorescent protein tethering (8,9).
RNA localization has been studied extensively in Xenopus laevis oocytes, where RNAs are localized during oogenesis and underlie patterning along the animal-vegetal axis (3–6, 10–18). The Xenopus oocyte offers several significant advantages for studies of RNA transport. First, oocytes are easily obtained through non-lethal surgery. Each surgery can yield thousands of oocytes, making the system amenable to biochemical analyses. Second, oocytes are large in size, easily visible in detail under standard light microscopes, offering facile microinjection of RNA, proteins, DNA, and antibodies, which can be targeted into the nucleus or cytoplasm. Third, isolated oocytes are amenable to culture outside of the frog (19,20). However, one disadvantage is increasing opacity as yolk protein accumulates during oogenesis, complicating imaging approaches. Here we describe a method of visualizing RNA localization in Xenopus oocytes that overcomes this issue while providing striking, high-resolution images of in vivo RNA transport.
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.
1To calculate RNA yield, first determine cpm in “input” (see 3.1.9) and “incorporated” (see 3.1.12) samples using a standard scintillation counter (note that the “input” represents 1% of the sample, while the “incorporated” represents 10% of the sample).
2Problems with low RNA yield could be due to one or more of the following issues:
3The steps below can help to prevent problems with needle clogging during microinjection:
4Problems with oocyte viability generally stem from extended collagenase treatment during oocyte isolation or non-sterile conditions during oocyte culture. The following precautions are necessary:
5The 10x MEM stock is good for several months as long as it remains colorless; store at room temperature wrapped in aluminum foil. 20 minutes is the minimum time for fixation; however, we can also fix for several hours without negatively affecting oocyte quality.
6Autofluorescence of yolk proteins in the oocyte places limitations on the choice of fluorescent nucleotides and secondary antibodies. If possible, fluorescent nucleotides and secondary antibodies giving emission at longer wavelengths (e.g., 546 nm, 633 nm) should be used, as autofluorescence is significant at shorter wavelengths (e.g., 488 nm).
7Problems with signal detection can occur, and may arise from a number of sources:
8Additional imaging tips: We generally image with a fairly open pinhole (>1 Airy Unit), as fluorescence intensity can be quite weak. However, results may vary from batch to batch of ooytes. Additionally, some autofluorescent subcellular structures may be visualized in the 488 nm (green channel), which can be useful for orienting oocytes along the animal/vegetal axis.