Ring Culture Procedure Modified After ref [12 with Preincubation in Oligos

Part 1 is carried out in the dish (see Subheading 2., item 12) with a black area on the bottom, in a layer of PC saline covering the yolks, at between 25 and 30°C (where they rapidly cool to 20°C; see Notes 3 and 4). Initially, process 6 yolks maximum/25-cm diameter dish, though with skill (speed in this case), 15 or more can be set up from such a dish. This is a naked-eye operation with good overhead lighting, but many workers could also use an engineers "anglepoise" lamp, which is in a ring around a large low-power magnifying lens that can be pulled over saline surface or bench.

1. Wipe eggs, straight from 38.5°C incubator, with 70% ethanol. Leave with blunt end upward for 5 min before taking first one. Crack carefully around with taps of butt end of big forceps, then insert tips parallel with the shell, removing a "cap" of shell from the blunt end of one-fourth to one-third the depth of the egg. Using the edge of the shell and the blunt-ridged forceps, separate the yolk from its hammock of thick chalazal albumen and then discard the latter by tipping out of shell. The "thin" albumen for the culture medium can then be removed with the blunt-mouthed pipet and pooled, from several eggs, into a small flask. Then tilt the yolk carefully into the saline, and turn it to float blastoderm uppermost. When all yolks are in the dish, go around them clearing off as much as possible of remaining jelly-like albumen coating their upper hemispheres using scissors and curved blunt-toothed forceps. The more of this removed at this stage (preferably right out of the dish), the better.

2. Line up yolks round edge of dish, and work by bringing each yolk opposite you in succession by smoothly turning the dish. Using the (washed or different!) big forceps again, place a watch glass and ring assembly under the saline on the black background in middle of dish, and remove the ring to one side. Using one of the specially blunt-tipped no. 5 forceps and the straight scissors, cut around yolk slightly above the "equator" to give a disk of vitelline membrane with blastoderm very near its center and still toward the saline surface. Cut toward forceps as they rotate yolk toward you. With a firm and steady pulling motion by both pairs of small blunt forceps, peel the membrane disk by its edges away from you, and off the yolk onto the watchglass, inner side uppermost. Centralize disk on watchglass, and remove major folds. With skill, this is done with movements of the ring itself while held in the forceps, just before it is placed polished face downward to pin the membrane, with blastoderm near its center, in the center of watchglass. This movement should be done within a few seconds, since a thin albumen layer on the back of the membrane otherwise contracts it to near ring size.

Develop skill in using the forceps to ensure that at the beginning of the peeling movement, yolk separates from membrane to leave a rather "clean" membrane, but with attached blastoderm and a peripheral skirt of yolk in the middle. Also, younger blastoderms are more likely to remain adherent to membrane if the use of a two-handed pulling technique ensures that folding and tension lines across them are minimal. Do not grip membrane too hard or very near its edges, or it will tear there. Temperature of the saline, age of eggs, stage of egg incubation, and length of time in saline before performing this step all affect the likelihood of blastoderm staying on membrane and yolk separating cleanly. Only experience with each stage of blastoderm can help optimize this. If much yolk remains on membranes, it is best to blow it off with pipets at his stage (hence the 6 eggs/dish recommended for starting!). See steps 5 or 6 if blastoderm has stayed on yolk or floated off into saline, but also proceed to steps 3 and 4 with each membrane/ring assembly as is needed for each blastoderm to be cultured. Speed makes for success of the whole culture experiment, since the more briefly each yolk/blastoderm is in saline only, the more likely it is to stay on membrane for transfer and the better its subsequent resumption of development.

3. Preferably (for quickness) without moving to microscope at this stage, even out membrane wrinkles or folds, clear away spare albumen strands and yolk, and stretch the blastoderm slightly, but evenly on membrane within the ring, maximizing the amount of membrane disk outside the ring. This is done with the blunt fine forceps and the special curved fine Pasteur pipet (but remember, no membrane holes!). With small movements, points on the ring can be lifted slightly while membrane is pulled beneath it, and with skill, many small, uneven membrane disks can be "rescued" at this point. Start by cutting overlarge disks, and with practice they can be made smaller and neater, since having just the necessary membrane to overwrap the ring (see Part 2) is advantageous. If preferred, this step can be done after "lifting out" of each watchglass/ring assembly into its culture dish as follows, and under the microscope.

4. Lift out the entire assembly with the large forceps, and keeping it almost horizontal but draining some saline from one edge, place in the Petri dish on the filter paper ring. By pipeting saline off from around the ring, then gently into the ring, and then lifting the ring gently at one point, it can be assured that the blastoderm itself sinks well under the saline meniscus and is not bulging up to touch this, which may disrupt its structure. Then, partially replace the PC saline with 1:1 Liebovitz TCM:Hank's BSS mixture inside the ring. Bring each blastoderm from one dish of yolks or, for each experiment, to this stage before proceeding. The provision of TCM:BSS at this point, an addition to the original New culture technique, ensures in our experience that streak stage 4 and even younger streak and prestreak stage blastoderms can wait at room temperature (around 20°C rather than warmer) for up to 3 h or longer without loss of capacity to develop onward. Even so, in experiments involving the younger embryos, waiting times before Part 2 below should be shorter if possible. Proceed to Part 2.

5. As a parallel step at this point, if blastoderms that came off membranes at step 2 are to be used in the experiment (virtually obligatory in work on very young stages and, on some days, with most streak stages), these should be removed individually at step 2 by a blunt-mouth pipet from the PC saline dish, and spread out in a 3-mm deep layer of TCM:BSS in a Petri dish at room temperature. These seem to stay in best condition if manipulated to be epiblast (former membrane-facing) side up in the medium. They can then join blastoderms removed manually from membranes (Part 2, step 2), and should be in equivalent physiological condition.

6. In addition, the very youngest blastoderms (prestreak to stage 2) sometimes stay on the yolk surface itself at step 2, and can be removed and stored in a way that optimally preserves their developmental potential. This is again done for each individual after bringing its membrane setup to the end of step 4, so that blastoderms that have stayed behind on the yolk are not left in the large PC saline dish for more than a few minutes each. A low-power engineer's magnifier lamp or equivalent over the dish is very desirable for this. The blastoderm is cut out, together with a shallow yolk floor underlying the subgerminal space, by excavating movements with two pairs of the no. 5 forceps. One fine-tipped pair and one as used in holding vitelline membrane are optimal, and care is necessary to cut just outside the true cellular border of blastoderms, usually visible as a white line on pale yellow yolk. This whole is then transferred to a plastic Petri dish in a layer of TCM:BSS using the blunt-mouthed pipet, and stored blastoderm epiblast uppermost as in step 5. This and step 5 represent the second-best storage condition out of the egg at room temperature, after the condition described in step 4 above or, best of all, the end of Part 2, step 1. About 20 such blastoderms can be stored/60-mm plastic dish in 5 mL of medium, but these very young ones should only be separated from their yolky masses close to the time of their incubation in oligos, and so forth, or their direct transfer back into completed ring cultures. This separation can be done with one pair of fine no. 5s and the flattened mounted needle, by inverting, cutting into the upturned subgerminal cavity through the yolk floor with cross-shaped cuts until the white hypoblast cellular surface is clearly seen, and peeling off the yolky quadrants. Cooling from room temperature in a 4°C refrigerator for a few minutes only will bring a dish of these blastoderms to a condition where the yolk separates from the cells with least likelihood of damage.

Part 2 is completed with each culture under the lowest power of the dissecting microscope so that the entire ring can be viewed at once. Because depth perception is much better, incident lighting rather than transillumination is best for this and all subsequent operations (except for examining development itself without terminating the cultures).

1. The pairs of small blunt forceps are used to complete the process of centralizing the blastoderm, evenly stretching the membrane and then folding it up to wrap over the (rougher) upper face of the ring so as to form a potential seal all around the ring bottom. To do this, saline must first be pipeted from inside and outside the ring until the top face is free of it. Forceps tips are best kept near the same point, opposite your body on the ring periphery, and the whole watchglass or Petri dish rotated as you work, rather than the hands following the changing angle of the ring edge. Initially, the ring alone is lifted slightly while the other forceps wraps a region of membrane completely over the top face from the outside. Subsequently, one forceps grasps membrane-ring-membrane at the point where wrapping over has just been done, and lifts it very slightly off the watchglass, while the other grasps the edge of the membrane and slides it up the outside of the ring, stretching it slightly along the ring while pulling it over. By adjusting the degree of this stretch at each location, the blastoderm can with practice be centralized within the ring. The procedure is quickly learned, with fewest punctured membranes, when the wrists are supported and relaxed, and the ring turned rather than "following" its curvature with your hands. Once a hole has been made however small, in the membrane inside the ring or along the lower half of the outer ring wall (further up will be okay with luck), then abandon and start another assembly, since the ring will inevitably flood with albumen medium later on. Hence the importance of blunt smoothed forceps and totally smoothed pipet mouths. Remember that a certain number of spare membrane-ring setups (from casualty blastoderms) act as backups for the transfer of good blastoderms from holed membranes! If the timing of an experiment allows for it, or if two operators are working together, the optimal condition for holding blastoderms while accumulating numbers for oligo treatment is to then half-replace the fluid under the lightly stretched vitelline membrane with albumen culture medium (see Subheading 3.3., step 3 below for use of special-angled pipet), keeping a shallow layer of TCM:BSS above the blastoderm, but accumulation at step 4 of Part 1 is normally more practical and adequate.

2. Removal of blastoderms from vitelline membrane: The inside of the ring should now be half-full of TCM + saline, and sufficient albumen medium should lie beneath the slightly stretched membrane to form a cushion. Attempts to remove well-adherent blastoderms without first stretching membranes over rings are more likely to result in membrane puncture, so that blastoderm needs a new membrane setup. Overlying saline should be clear enough to allow good visibility without reflection from the incident light. Now is the time to remove pieces of the hypoblast/endoderm if this is to be done. We think this offers no advantage in cases of genes initially activated in epiblast, even if the cells concerned are presumptive mesoderm, but it may improve access of oligos to the already mesodermal cells of gastrulating stages. Hypoblast is a papery thin epithelium, closely adherent to underlying cells near the streak midline, but less so more peripherally. Good oblique incident light and the smooth, flat-tipped mounted needle are necessary to get underneath it, and loosen and remove a piece in the axial (usually the most relevant) region. This does not in itself affect subsequent development if the removed window is not too big and the streak well established (st. 3+ onward). Otherwise, it does cause developmental delay and a tendency to deficiency of anterior axial structures.

To take off tightly adherent blastoderms, the smooth flat needle is first used, working on the area opaca periphery nearest your body and bringing new regions to this location by rotating the whole assembly toward the needle point. Experience is required to locate the exact edge where the cellular epiblast is migrating on the membrane, in examples of different ages removed from yolks under different temperature conditions (affecting the amount of yolky material that is left overlying this junction). A very small wound must initially be made to insert the needle tip, almost flat, into the space between blastoderm and membrane without puncturing the latter. Then, using a sideways movement toward the periphery, unseal the first bit of this from the membrane. It is always worth quickly looking for a place to begin, however small, where the periphery has come unstuck by itself. On "good" days, most blastoderms up to headfold stages have at least one such place, and life is quicker because from this point on, the fine smoothed jeweler's forceps can be inserted in the gap. The whole periphery can then be unsealed with a quick succession of small sideways movements of the closed forceps tips, with much less chance of damage to blastoderm or membrane. Blastoderms freed in this way should be folded in two inside the ring and removed to be stored flat in dishes of TCM:BSS as in Subheading 3.1., Part 1, steps 5 and 6. Use the medium-mouthed pipet. Using the wide-mouth pipet in the restricted fluid volume inside the ring is hard to control without disruption of the blastoderm and/or punching a hole in the membrane.

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