カエルとサンショウウオのリビング卵母細胞からジャイアントランプブラシ染色体の分離

The overall goal of this procedure is to isolate the giant nucleus, or germinal vesicle, from an amphibian oocyte, and to spread the lampbrush chromosomes, and other nuclear organelles, on a microscope slide for immunostaining, in citu hybridization, and other molecular techniques. This method can help answer key questions about the basic structure of chromosomes, nucleoli, and other nuclear organelles as well as molecular details of transcription, splicing, and RNA export. The main advantage of this technique is the ease with which chromosome structure and transcription can be examined by a conventional light microscopy, using immunofluorescence, in citu hybridization, and other molecular techniques.

Demonstrating the procedure will be Dr.Zehra Nizami, a postdoc from my laboratory. After preparing all of the required solutions, assemble well slides, composed of subbed, glass slides, to which plastic wells have been attached with paraffin, as discussed in the text protocol. Then gather ovarian tissue previously collected from an adult, female frog or a salamander, and fill a small Petri dish with oocyte culture medium.

Proceed to place a portion of the isolated tissue, one containing approximately 10 to 20 oocytes, into this plate. Next, identify a single oocyte and use two pairs of jewelers forceps to sever the stalk that connects it to the ovary wall. Once it is freed, transfer the oocyte to a new Petri dish containing germinal vesicle, or GV, isolation medium.

Under low magnification, locate the animal pole of the oocyte, which can be identified by its dark coloration. Then use forceps to poke a large hole near the animal pole and proceed to gently squeeze out the transparent oocyte nucleus, also referred to as the GV.Afterwards, roll the GV away from any yolk that was also extruded. To remove yolk that remains tightly adhered, draw the nucleus in and out of a pipette with a tip having a diameter slightly larger than that of the GV.Following the collection of a frog GV, immediately transfer it to dispersal medium in order to prevent nuclear hardening that would interfere with subsequent steps.

Next, with two pairs of forceps, grasp the nuclear envelope near the top of the GV, taking care not to press down. While still gripping the envelope, proceed to pull the two forceps apart and expect the contents of the nucleus to pop out. To collect these contents, first prepare an adjustable 20 microliter pipette with a cut tip as described in the text protocol.

Then, from the dish, proceed to draw up five microliters of dispersal liquid followed by five microliters of the solution with the GV contents. Once these contents have been collected, transfer them to a well slide filled with dispersal solution. Ensure that the liquid in the slide has a convex surface so as to avoid the trapping of bubbles in subsequent steps.

Afterwards, position an 18 millimeter cover slip over the well and place the assembled slide into a moist chamber for 10 to 60 minutes. During this time, expect the nuclear gel to slowly disperse, so that the chromosomes and nuclear organelles come to lie on the surface of the glass. After removing a nucleus from a salamander oocyte, leave it in the isolation medium.

Note that salamander GVs, unlike those of the frog species used here, will not unduly harden in this solution. Remove the nuclear envelope from the salamander GV using the same forceps-based technique as described for frogs. Next, pick up the slightly gelled nuclear contents with a pipette having a cut tip, and transfer the gel to a Petri dish containing dispersal solution.

Quickly rinse the pipette in the dispersal solution, and use it to collect the nuclear gel. Then introduce the gel into a well slide, add a cover slip, and allow the gel to disperse as previously described. Then use phase-contrast to view the nuclear contents, and identify the lampbrush chromosomes, abbreviated as LBCs, by their large size and paired, lateral loops.

After confirming that the LBCs are unbroken, prepare the slide for centrifugation by placing a piece of filter paper on the cover slip. Then press down to remove excess liquid from the chamber. Next, add petroleum jelly to each edge of the cover slip.

Heat a metal rod to approximately 70 to 80 degrees Celsius and use it to melt the jelly and seal the cover slip onto the underlying plastic. Repeat this procedure for other slides with LBCs of good quality. Once all the cover slips have been affixed, position the slides in carriers for centrifugation and ensure that opposite carriers are balanced.

Using the slow start function, centrifuge the slides as described in the text using custom-built carriers. Alternatively, a standard tabletop centrifuge can be used where slides are taped to a plate and spun. To prepare the nuclear contents for immunostaining or in citu hybridization, first wipe away the temporary Vaseline seal with a razor blade, and then place the slide into the rack of a standard slide-staining dish.

Next, add enough paraformaldehyde to cover the slides. Upon submergence, use blunt forceps to remove and discard each cover slip. Leave the slides in fixative for 15 to 30 minutes.

Following fixation, place a slide into a staining dish containing ice cold PBS. Then insert a sharp razor blade at the edge of the plastic square, and pry this plastic well loose. Once this process has been repeated for all the slides, proceed with staining as discussed in the text protocol.

Shown in this dark-field illumination image are the nuclear contents from a single axolotl GV in prophase of the first meiotic division, collected and prepared using the described protocol. 14 large, paired LBCs are apparent, as are amplified nucleoli. Additional staining of this preparation, with an antibody against phosporylted RNA polymerase II, shown here in green, reveals the locations of histone locus bodies and also provides insight into the robust, transcriptional activity of these oocyte chromosomes.

Higher magnification of a single axolotl LBC, in this instance one that has been stained green for RNA polymerase and blue for DNA, demonstrates the most unique morphological feature of these chromosomes. Hundreds of paired lateral loops, each of which represents at least one transcription unit that actively synthesizes RNA. Comparison with a similarly stained frog LBC, imaged at the same magnification, emphasizes the enormous size difference between frog and salamander chromosomes, a difference that correlates with the total DNA content of the genomes of the species used.

However, even in the frog chromosome, the individual transcription units can be seen as loops of chromatin projecting laterally from the chromosome axis, as shown in this higher magnification image. Oocytes of fish, birds, reptiles, and some invertebrates also contain giant lampbrush chromosomes. Remarkably, lampbrush chromosomes are generated when sperm heads from other organisms, including humans, are injected into the amphibian germinal vesicle.

Once the technique is mastered, a batch of AGV spreads can be prepared in one hour, and then centrifuged and fixed within another one to two hours. After watching this video, you should have a good understanding of how to isolate a GV and spread the lampbrush chromosomes for further study using molecular probes.