JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

Published: December 5th, 2016

DOI:

10.3791/54103

1Department of Embryology, Carnegie Institution for Science

We present simple techniques for isolating giant transcriptionally active lampbrush chromosomes from living oocytes of frogs and salamanders. We describe how to observe these chromosomes "alive" by phase contrast or differential interference contrast, and how to fix them for fluorescent in situ hybridization or immunofluorescent staining.

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and salamanders. Individual LBCs can be up to 1 mm in length and they reside in a gigantic nucleus, itself up to 0.5 mm in diameter. The large size of the chromosomes permits unparalleled observations of active genes by light optical microscopy, but at the same time special techniques are required for isolating the nucleus, removing the nuclear envelope, and spreading the chromosomes on a microscope slide. The oocyte nucleus, also called the germinal vesicle (GV), is isolated in a medium that allows partial gelling of the nuclear actin and preserves the delicate structure of the LBCs. This step is carried out manually under a dissecting microscope using jeweler's forceps. Next, the nuclear envelope is removed, again manually with jeweler's forceps. The nuclear contents are quickly transferred to a medium that disperses the actin gel and allows the undamaged LBCs to settle onto a microscope slide. At this point the LBCs and other nuclear organelles can be viewed by phase contrast or differential interference contrast microscopy, although finer details are obscured by Brownian motion. For high resolution microscopical observation or molecular analysis, the whole preparation is centrifuged to attach the delicate LBCs firmly to the slide. A brief fixation in paraformaldehyde is then followed by immunofluorescent staining or in situ hybridization. LBCs are in a transcriptionally active state and their enormous size permits molecular analysis at the individual gene level using confocal or super-resolution microscopy.

Most vertebrates, with the notable exception of marsupials and placental mammals, produce large yolky eggs. Despite their sometimes enormous size, these eggs are single cells that reach their final dimension while still in the ovary of the female. Ovarian eggs are called oocytes and each typically contains a single giant nucleus, known since the early 19th century as the germinal vesicle or simply GV.1 Oocytes of the common laboratory frogs, Xenopus laevis and Xenopus tropicalis, reach a maximal diameter of 1.2 mm and 0.8 mm respectively (Figure 1). The GVs from mature oocytes of these two frogs are 0.3 - 0.4 mm....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

General information about frogs and salamanders, as well as sources of animals, can be found at the following websites: Xenbase (http://www.xenbase.org) and Sal-Site (http://www.ambystoma.org). This protocol follows the animal care guidelines of the Department of Embryology of the Carnegie Institution for Science.

1. Solutions

  1. Make 10 L "frog water": Add 10 mL of 1 M CaCl2 and 10 mL of 1 M NaHCO3 to 10 L deionized or dechlorinated H2O with s.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

To examine giant lampbrush chromosomes one begins by isolating oocytes from a frog or salamander. Figure 1 shows a group of mature oocytes in a buffered saline solution after removal from the ovary of the frog, Xenopus. Such oocytes remain in good condition for days at room temperature. The nucleus (or germinal vesicle) is then removed from an oocyte with jeweler's forceps, either in a saline solution (Figure 2) or in oil (Figure 3

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The first observations of "living" LBCs from hand-isolated GVs of frogs and salamanders were made nearly 80 years ago by the American biologist William Duryee,19 before the introduction of phase contrast and DIC microscopy, before fluorescent immunostaining, and before FISH. The advantages of LBCs for investigating details of chromosome structure and transcription at the individual gene level required development of techniques to attach the LBCs to glass slides, to fix them in a lifelike manner, and to.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM33397. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.G.G. is American Cancer Society Professor of Developmental Genetics.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Paraformaldehyde (reagent grade, crystalline) Sigma-Aldrich P6148-500G Despite warnings in many protocols, a concentrated solution can be stored indefinitely at room temperature
Ethyl 3-aminobenzoate methanesulfonate Sigma-Aldrich A5040-100G Sometimes referred to as MS-222
Ethicon RB-1 1/2 circle taper point 3-0 sutures VWR  95057-000
Paraplast (paraffin wax) Sigma-Aldrich P3558-1KG
p-Phenylenediamine Sigma-Aldrich P6001
Gelatin Grocery Store Commercial Knox gelatin works fine
ProLong Gold antifade mountant ThermoFisher Scientific P10144
Gold Seal cover glass  22 x 22 mm #1 1/2 (0.16-0.19 mm thick) Electron Microscopy Sciences 63786-01 These coverslips are the recommended thickness for superresolution microscopy
Dumont forceps #5 Electron Microscopy Sciences 72700-D http://www.emsdiasum.com/microscopy/products/tweezers/dumont_positive_action.aspx
Paraffin oil (light)  EMD Chemicals PX0047-1 For isolating GVs in oil
Adhesive in situ PCR and hybridization chambers (25 µl). BioRad Frame-Seal Slide Chambers #SLF0201 http://www.bio-rad.com/en-us/sku/slf0201-frame-seal-slide-chambers
Silicone isolators Grace-Biolabs select from catalog link http://www.gracebio.com/life-science-products/microfluidics/silicone-isolators.html
Coplin jars and staining dishes Electron Microscopy Sciences select from catalog link http://www.emsdiasum.com/microscopy/products/histology/staining.aspx

  1. Purkinje, J. E. . Symbolae ad ovi avium historiam ante incubationem. , (1830).
  2. Rückert, J. E. Zur Entwickelungsgeschichte des Ovarialeies bei Selachiern. Anat. Anz. 7, 107-158 (1892).
  3. Callan, H. G. . Lampbrush Chromosomes. 36, (1986).
  4. Gall, J. G., Callan, H. G., Wu, Z., Murphy, C., Kay, B. K., Peng, H. B. Lampbrush chromosomes. Xenopus laevis: Practical Uses in Cell and Molecular Biology. Vol. 36 Methods in Cell Biology , 149-166 (1991).
  5. Gall, J. G., Wu, Z. Examining the contents of isolated Xenopus germinal vesicles. Methods. 51, 45-51 (2010).
  6. Penrad-Mobayed, M., Kanhoush, R., Perrin, C. Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes. Methods. 51, 37-44 (2010).
  7. Morgan, G. T. Working with oocyte nuclei: cytological preparations of active chromatin and nuclear bodies from amphibian germinal vesicles. Methods Mol. Biol. 463, 55-66 (2008).
  8. Paine, P. L., Johnson, M. E., Lau, Y. T., Tluczek, L. J., Miller, D. S. The oocyte nucleus isolated in oil retains in vivo structure and functions. Biotechniques. 13, 238-246 (1992).
  9. Handwerger, K. E., Cordero, J. A., Gall, J. G. Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure. Mol Biol Cell. 16, 202-211 (2005).
  10. Patel, S., Novikova, N., Beenders, B., Austin, C., Bellini, M. Live images of RNA polymerase II transcription units. Chromosome Res. 16, 223-232 (2008).
  11. Gall, J. G. Octagonal nuclear pores. J Cell Biol. 32, 391-399 (1967).
  12. Löschberger, A., et al. Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J Cell Sci. 125, 570-575 (2012).
  13. Gottfert, F., et al. Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. Biophys J. 105, L01-L03 (2013).
  14. Wallace, R. A., Jared, D. W., Dumont, J. N., Sega, M. W. Protein incorporation by isolated amphibian oocytes: III. Optimum incubation conditions. J. Exp. Zool. 184, 321-333 (1973).
  15. Bridger, J., Spector, D. L., Goldman, R. D., Leinwand, L. A. Fluorescence in situhybridization to DNA. Cells: A Laboratory Manual. 3, 111.111-111.136 (1998).
  16. Singer, R. H., Spector, D. L., Goldman, R. D., Leinwand, L. A. In situ hybridization to RNA. Cells: A Laboratory Manual. 3, 111-116 (1998).
  17. Gardner, E. J., Nizami, Z. F., Talbot, C. C., Gall, J. G. Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis. Genes Dev. 26, 2550-2559 (2012).
  18. Talhouarne, G. J., Gall, J. G. Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes. RNA. 20, 1476-1487 (2014).
  19. Duryee, W. R. Isolation of nuclei and non-mitotic chromosome pairs from frog eggs. Arch. Exp. Zellforsch. 19, 171-176 (1937).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved