Roller Bottle Culture After Oligo Treatment

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Our laboratory has recently reported successful culture of chick embryos in a version of the roller-tube method used for mammal development (13). This permits more extended normal development (30 +somites), probably because the extraembryonic blood-vascular supply becomes as profuse as that in ovo, whereas this does not occur in ring culture. We now have evidence that phosphorothioate oligos simply added to these cultures can maintain interference with the same genes that have been successfully treated by the modified New ring-culture method described in Subheadings 3.1.-3.3. above. However, probably for reasons connected with the oligo uptake lipofection procedure, the strongest sequence-specific effects are seen in roller-tube culture after oligo treatment as in Subheading 3.2., the static incubation in a shallow layer of protein-free medium. We therefore include a description of the current roller-bottle method. Such embryos develop resembling fish larvae in that they are linearly extended along the top of a "yolk-sac" derived from the extraembry-onic area pellucida and area vasculosa. Their two abnormalities are that normal torsion of the embryo does not develop and the heart tube displays dorso-ventral looping only, and that local failures of dorsal neural closure are liable to remain, even though CNS regional structure, neural crest emigration, and so forth, are normal. The latter feature, which could even offer an advantage for continued oligo treatment of the generative layer of the CNS, is seen just in individuals that were set up during the most intense folding movements of neural plate formation, rather than before or afterward.

Our current roller-culture medium is 100% Liebovitz TCM as above, plus 5 (when oligos are in medium) to 20% fetal calf serum. We already have evidence for necrosis, however, particularly in the notochord/floorplate region of the cross-section, in advanced embryos following growth in this medium. It is very likely that serum-free media, such as those developed for advanced chick organ culture (e.g., 18), will give even better and more extended development, since these and other authors have evidence for toxicity of serum to chick embryo structures. This offers the exciting possibility that half-life of phosphorothioate oligos in such cultures could be prolonged, since serum frequently contains exonucleases. Since we have not yet done the necessary testing ourselves, we draw attention to this, but do not detail the alternative media.

1. Remove the oligo incubation dish from incubator, and place on a warm bench or in a larger dish with a layer of warm water, thus avoiding cooling shock to blastoderms. Increase the depth of TCM:BSS to 5-6 mm with warmed medium, such that blastoderms of the ages in the dish can be folded in two without interference from the liquid surface.

2. Fold each blastoderm in two along the embryo's midline (see Fig. 3), endoderm side inward, so that the midline is slightly stretched out and folds in the area opaca are eliminated, and crimp together the inner faces of the area opaca (the yolky celled, peripheral ring of the blastoderm) at two or three pairs of opposing positions around the perimeter to maintain this folded configuration. This is best done with two pairs of the smoothed, relatively blunt, but well-fitting no. 5 forceps.

3. Holding the folded blastoderm by its curved periphery with forceps near one end of the axis, cut around the entire periphery with iridectomy scissors, just inside or outside the area opaca/ area pellucida junction, but avoiding damage to the poste-

Embryo Culture Roller

Fig. 3. Roller-bottle culture: At left is shown in plain view a blastoderm with somewhat older head process than that of Fig. 1. This is a good stage to commence with, although blastoderms down to stage 3 respond to the technique. At right is indicated the folding along the midline with epiblast outward, crimping with forceps at a few positions to stabilize the folding, and then cutting near the pellucida/opaca junction with iridectomy scissors. Below is sketched an embryo after approx 24 h of culture from such stages, showing how the extraembryonic disk, including its blood vascular system, develops as a "yolk-sac" with the embryo proper extended along the top.

Fig. 3. Roller-bottle culture: At left is shown in plain view a blastoderm with somewhat older head process than that of Fig. 1. This is a good stage to commence with, although blastoderms down to stage 3 respond to the technique. At right is indicated the folding along the midline with epiblast outward, crimping with forceps at a few positions to stabilize the folding, and then cutting near the pellucida/opaca junction with iridectomy scissors. Below is sketched an embryo after approx 24 h of culture from such stages, showing how the extraembryonic disk, including its blood vascular system, develops as a "yolk-sac" with the embryo proper extended along the top.

rior end of the streak or future tailbud. This creates a sealed, somewhat flattened lemon-shaped pocket that has the embryonic axis along one edge and the seal along the other. Step 2 and and this step are best done in succession on each blastoderm. The size of iridectomy scissors is obviously influential, as is the fact that younger blastoderms are relatively thicker and less stably foldable than older ones, but full-streak and even younger-staged blastoderms can be prepared and roller cultured in this way, as well as embryos of up to 10 somites at the start of culture. Cutting just outside the area opaca junction is probably optimal for prolonged culture, since the annular blood vessel normal to this region then develops fully. Folding hypoblast/endoderm out before cutting, giving a layer configuration equivalent to that in a mammal egg cylinder, does not result in normal development in the bird embryo.

4. Using the medium-mouth pipet, transfer such preparations in groups of up to three to the small plastic roller bottles described in Subheading 2., each containing 1 mL of the culture medium prewarmed (see introduction to this subsection). Lightly screw-seal caps and place on the inclined roller to rotate immediately. Present experience suggests that lower concentrations of oligos or oligo-

lipofection mixtures than those typically in static preincubation dishes are sufficient to maintain a level of antisense interference in the roller culture.

It appears that serum is hardly required for the first 15 h of culture from streak/ headfold stages, but is required for longer culture periods. Medium should be half-replaced every 12-15 h, using 20% serum for older embryos beyond 20 somites, for the longest culture. It is not yet known what the most advanced processes are in chick development whose genetic control can be investigated with antisense treatment of the whole embryo in this way.

Some aspects of final anatomy in these embryos are best seen after saline washing and under incident light, but before cutting off the extraembryonic sac, which leads to contraction of the unfixed tissue. Considerable fixation before this is cut away will prevent contraction of ventro-lateral parts of embryos and axial curling, which would otherwise make them less interpretable in whole-mount in situ and sectional series than embryos fixed from ring culture.

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