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

Summary

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.

Abstract

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.

Introduction

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 19^th 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....

Protocol

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 stirring.
2. Make 1 L 20% paraformaldehyde: Make 1 L of 4 mM Na₂CO₃. In a chemical hood add 200 g of powdered paraformalde....

Results

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

Discussion

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.......

Materials

List of materials used in this article

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 RT
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