The antisense effect in this work is relatively transient within the time scale of development (one of its potential analytical advantages), so that it works best if little developmental pause in real time occurs immediately following administration of oligos. Chick blastoderms at streak headfold stages are very susceptible to a delay in resumption of development even at normal incubation temperature, following experience of a rapid downward temperature shift. Ideally, therefore, the return to ring culture (and maybe even the whole procedure from the egg) would be at incubation temperature throughout. This is impracticable, but the local bench temperature, for the dish in which blastoderms have been incubated with oligos should be as high as possible during the period required for transfer of a set of them back to the culture setups. In particular, use of a larger dish as a warm water jacket to the "oligo dish" so that there is no downward temperature shock to blastoderms while awaiting transfer has led to the most rapid and normal resumption of development and the strongest relative antisense effects. Certainly, chilling of blastoderms at this stage may even prejudice the final result, and not merely the time schedule of subsequent "control" development.
1. The depth of TCM:BSS must be increased at this stage to facilitate pipet transfer (while preserving some of the oligo concentration around embryos). This added medium should be prewarmed to near-incubation temperature (see above). By gently oscillating the dish, blowing with a fine medium-filled pipet, or the use of forceps, it should be ensured that each blastoderm is individually prepared for the rapid pipet transfer of the following step by being free-floating, and not stuck to the plastic floor of the dish. This is much easier for those incubated "epiblast-up."
2. Lift the ring/membrane assembly at one point (with the small curved-toothed forceps) and withdrawing the remaining fluid from beneath it using the special bent, fine smooth-mouthed pipet. Rolling the ring around helps remove more of this underlying saline, and having the overlying space about one-third filled with TCM:BSS also helps by "pressing" saline out more thoroughly from beneath. The optimal pipet is so angled that its oblique smooth mouth lies horizontal and is easily placed beneath or (see later) into the space around the inner edge of the ring.
3. Place two successive Pasteur pipet bulbs-full of the albumen ring-culture medium beneath the membrane using the same pipet (no bubbles!) to total about 1.75 mL for a 25-mm diameter ring. In between the bulbs-full, blastoderms are replaced into the ring space in a minimal depth of protein-free medium, epiblast side down to the membrane as originally. Medium is added from the oligo dish to that within the ring space, so that when the blastoderm is delivered from the blunt or medium-mouth pipet, it can be spread over the central clear space on the slightly domed (convex) membrane using fine smooth forceps. Spreading is helped by the shal-lowness of the fluid layer at this position.
4. After the second bulb-full of albumen is beneath the membrane and the ring released, the meniscus overlying the membrane will, ideally, pin the blastoderm to its central position and allow it to spread, as medium is progressively withdrawn from around it with the same special pipet. In draining this space, concentrate on the gutter that develops next to the inner ring face as the membrane becomes convex from pressure of underlying albumen medium, and of course avoid damaging the blastoderm or sucking a hole in membrane. Cultures that are well drained before the start of incubation produce the best development. The above is an idealized, or even a personal description of what works best. As a procedural skill, this is the trickiest step to become adept at, but the learning process is nevertheless surprisingly rapid! Since any strands of nonfluid albumen can interfere with settling blastoderms on membranes, some prefer to use two, marked separate special pipets of the same design, one for albumen-pipeting steps and one for blastoderm-settling ones (i.e., under-membrane and above-membrane, respectively). Another way of optimizing the spread of the replaced blastoderm is by gentle stroking outward with the flat of the needle (Fig. 2).
For blastoderm transfer, the medium-mouthed pipet gives much greater control than the wide-mouthed, with the blastoderm folded or rolled up hypoblast side inward. It can then be unfolded, centralized, and if necessary turned hypoblast/endoderm side up with the fine-pointed forceps, using the area opaca edge, which can take local wounding. The saline depth is adjusted at this stage so that as soon as more is drained from the periphery, the blastoderm is abruptly pinned centrally by the meniscus onto the slightly convex membrane, rather than sliding to one side or the other. Underfilling with medium and draining to give full convexity is then completed. With a little more practice, as a final step, clean protein-free medium can be washed in and out under the blastoderm edges on a very small scale using fluttering movements on the bulb of the special pipet. With this technique, one can flatten out folds in the area opaca and centralize the flattened blastoderm on the dome of membrane, even removing any free lumps of yolk that sometimes are trapped beneath blastoderms. These particles, remaining in what will be the chick's equivalent of the mammal pro-amnion cavity, can interfere mechanically with closure of neural folds.
As in previous steps, most people find it best to learn to work at the part of the ring and blastoderm edge furthest from the body, and when necessary, rotating the whole assembly to bring new parts toward the hand holding the pipet or other instrument. The bend in the shaft and angling of the mouth of the special pipet is to facilitate all the steps of ring culturing, repositioning blastoderms, and draining the space within the ring.
5. The filter paper should always be saturated with water (or the saline) to preserve 100% humidity. Development can be observed from the ventral aspect in reasonably clean ring cultures. After about 18 h, if further culture is needed, they are redrained above and "fed" with a small amount of more medium beneath to preserve the membrane convexity and air/fluid interface, and the filter paper resaturated for humidity. Blastoderms set up in such cultures at streak stages will develop for up to 30 h, though with increasing mechanical compression of the head and heart region, and deficiency in the normal blood supply. In reality, around the 18 somite stage is the outer monitoring point for effects of oligos within this culturing system, and the most suitable genes for study with it are those that are required for normal expression of the basic body plan (outline of the CNS anatomy, heart, and first 10 somites).
For full and final examination (fixation for histology, in situ, and so forth), the ring should be flooded with warm PC saline, starting by dripping directly onto the embryo so that it remains intact below the meniscus rather than being lifted onto the fluid surface. The entire blastoderm is then peeled off the membrane using needle and/or fine forceps. Transfer by pipet to a saline wash removes spare protein and solid particles from the structure, greatly improving quality of specimens for whole-mount in situ or structural histology.
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