Laboratory of Chromosome Structure and Function

Protocols

 

Methods for making lampbrush chromosome preparations from oocytes of birds

Lampbrush chromosomes can be isolated manually employing the basic techniques developed for amphibians (see Callan, 1986; Macgregor and Varley, 1988) suitably modified for oocytes of birds (see Kropotova and Gaginskaya, 1984; Solovei et al., 1993).

Removal of the nucleus from the oocyte

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Remove an entire ovary from the bird, discarding all oocytes and eggs of more than about 5 mm diameter, blot it dry on a clean filter paper and place it in a sealed glass container with a piece of filter paper moistened with a few drops of 5:1 medium.

Keep the ovary on ice while working.

 

The size of oocyte size suitable for lampbrush dissection varies in different species. Use oocytes of 1 -2.5 mm from chicken and turkey, 0,5-0,75 mm from Japanese quail, 0,5-1,5 mm from pigeon and 0,25-0,75 mm from sparrow and chaffinch.

 

 

Dissect off and transfer individual oocytes of the required sizes to an embryo cup containing the 5:1 medium.
If the entire ovary is very small, as, for example, in chaffinch, place a piece of ovary in the embryo cup and separate the oocytes afterwards.

 

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Embryo cup with nuclear isolation medium

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Do not attempt to remove any of the follicular envelope tissue from around the oocyte.

 

 

For isolation of the oocyte nucleus you need two very sharp tungsten needles and a pair of fine (No5) steel watchmaker's forceps with points that meet precisely.

 

 

Usually, the oocyte nucleus (germinal vesicle) is clearly visible through follicular envelope as a transparent spot. However in smaller oocytes the nucleus is often situated deeply in the centre of the oocyte and cannot be seen.

 

Watch the nucleus carefully during the procedure, since if it comes out of the oocyte amidst a mass of yolky cytoplasm without you seeing it, it will be much harder to find subsequently.

It is important to keep the nucleus in the field of vision for a successful nuclear isolation

Keep watching the nucleus as it emerges from the oocyte

When you have made a “smile-like” hole in the oocyte envelope with the hook-like needle, pull the needle out of the egg and force the nucleus to leave the oocyte, pushing it gently with the same needle.

 

When the nucleus is out of the egg clean the adherent yolk from it using the two needles and then by picking it up in a very fine bored Pasteur pipette and gently pumping it in and out of the pipette.

Make your pipettes as directed in the protocol for amphibian material

Light spot = nucleus

Some adherent yolk, but usable

Use needles to clean yolk particles from the nucleus

 

 

Another embryo cup with chromosome isolation medium

 


Removal of chromosomes from the isolated nucleus

Chamber with chromosome isolation medium

Lampbrush ”observation chambers” are made from ordinary histological slides. Cut a slide to give a glass square of 24x24 mm and bore four 4 mm diameter holes in it. Then stick this glass square to an ordinary slide with an excess of rubber cement to make a perfect seal over the whole area of the square. Use enough cement for it to spread out over the bottoms of the wells created by the 4 holes. Use these slides within one day or keep them at +4o C; but do not keep them longer than one week in any circumstances. Do not attempt to remove the rubber cement from the bottoms of the 4 wells until you are ready to use the slide: the excess cement keeps bottoms of the wells absolutely clean and free from dust. When you are ready to use the slide, remove the cement from the bottoms of all the chambers by simply peeling it off with a pair of fine forceps.

As quickly as possible after you have removed the nucleus from the oocyte and cleaned off adherent yolk, transfer it by Pasteur pipette to the chromosome isolation medium (see below) in one of the 4 wells in the special slide.

Use enough medium in the chamber to create a very slight upward curving meniscus.

 

In order remove the nuclear membrane from the oocyte nucleus and spill out the chromosomes into the well chamber you will need two newly sharpened and very fine tungsten needles one of which should be slightly bent towards the tip. Raise the magnification of the dissecting microscope to a level at which you can comfortably see what you are doing with the nucleus.

Hold the nucleus by this needle

Use the straight needle to press carefully against one side of the nucleus without any damaging the nuclear membrane.

 

With the curved needle make a hole in the GV envelope nearby the point of the first needle.

 

 

Now tear off the nuclear envelope.
This can be done in different ways depending largely on the particularly peculiarities of the oocytes that you are working with, which will vary from one bird to another.

Nuclear contents

Nuclear membrane

The ideal ball of nucleoplasm

Pieces of nuclear membrane

 

After the nuclear contents have been deposited on the bottom of the well chamber, leave them to disperse for about 30 min at +4o C. Then the cambers should be centrifuged at 3000 g for 15 min in a cooled centrifuge. It is not normally necessary to cover the well chambers with coverslips, as specified in the amphibian protocols.

After centrifugation, fix the preparation in 2% formaldehyde for 2 min and postfixed in 70% methanol for several hours or overnight. Mark the locations of the well chambers on the microscope slide with a diamond on the slide back before removing the holed square of glass from the slide with a razor.
Chromosomes for FISH or PRINS should be dehydrated in 100% methanol, air dried and kept at -20o C. Preparations for immunostaining must be kept in 70% methanol at +4o C before using it is recommended that preparations not be stored in methanol for longer than is absolutely necessary.
You may use ethanol instead of methanol for nucleic acid investigation but not for immunocytochemistry.

Solutions:

5:1 medium for nuclear isolation

83 mM KCl
17 mM NaCl
6,5 mM Na2HPO4
3,5 mM NaH2PO4 (KH2PO4)
pH 7,0

Chromosome isolation medium

5:1 medium diluted in 4 times
0,25 mM MgCl2
0,1% formaldehyde

2% formaldehyde

5:1 medium diluted in 4 times
2% formaldehyde

 

 

References:
Callan HG (1986)
Kropotova EV, Gaginskaya ER (1984)
Macgregor HC, Varley JM (1988)
Solovey I, Gaginskaya E, Hutchison N, Macgregor H (1993)

Compiled by E. Kroptova, I. Solovei, A. Safitdinova and E. Gaginskaya.

LBCs_RNA_PolII

Lampbrush chromosomes microsurgically isolated form Japanese quail oocyte. Immunofluorescent detection of elongating form of RNA polymerase II (green signal) on the lateral loops of lampbrush chromosomes. Scale bar - 10 mkm.

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