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Traditional Chinese Medicine

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1. Pannett and Compton (PC) saline for bird embryos (original ref. given in 12) (PC saline). If filter-sterilized or autoclaved in the two parts when making up, this can be stored at room temperature for months. PC saline is needed in liter quantities per experiment dealing with 10-20 chick eggs, 500-mL bottles are thus convenient.

2. Hank's Balanced Salt Solution (BSS), made up with 0.1X the originally specified concentration of divalent cations CaMg2+. Use of the tissue-culture pH indicator dye facilitates important monitoring for freedom from the highly alkaline ovalbu-min before administration of P-oligos. Made up sterile as for PC saline, this is best stored in 20-mL universals of which 1/typical experiment will be required.

3. Liebovitz L-15 air-buffered tissue-culture medium without antibiotics, but with glutamine (TCM): Gibco-BRL (Paisley, Scotland). Best aliquoted into 10-mL tubes and stored at 4°C (shelf life 3 mo+ if true sterility maintained). One to two tubes needed per experiment.

4. Gentamycin as antibiotic (Sigma, Poole, UK): Stable for months at 4°C as a stock at 10 mg/mL (x200 working concentration) in sterile distilled water.

5. New ring-culture medium: Eight parts of the thin albumen from fresh eggs (avoid any nonfluid albumen—see Subheading 3. for opening of eggs): 1 part TCM: 1 part HBSS: 50 |g/mL Gentamycin. With good lab practice (i.e., washing all implements, containers, and surfaces in copious plain water between sessions), use of antibiotics should ideally be superfluous for chick embryo culture on the open bench because of the bacteriostatic properties of lysozyme in albumen. Their addition saves frustration in overcrowded multipurpose lab spaces, however. Such newly prepared medium can be kept at 4°C for 2-3 d in glass or tissue-culture plastic, but is normally prepared fresh for each experiment, 3 mL/planned ring culture should be available.

6. S-Oligos: Phosphorothioated, HPLC-purified and sequence-certified DNA-oligos, normally 15-mer. The most consistent, quickest, and best-value commer cial supplier of these is currently the U.S. Company, Oligos Etc. Inc., whose product takes up in sterile distilled water immediately with almost no residue, for use with no further purification. The "1-|im" synthesis scale normally yields 2+ mg of a 15-mer S-oligo, enough for 10 single-oligo experiments as described in the next section. Store according to manufacturer's instructions. We store 12.5-|L aliquots in 0.5-mL Eppendorf tubes at 2 mmol, and thaw from -70°C just before use. Such aliquots often become appreciably less effective after 5+ mo. Lyophilized storage should improve this.

7. Components of New ring cultures (see Fig. 1):

a. Glass watchglasses, approx 50 mm diameter, with as "deep" a curvature as possible. BDH (Poole, UK) has been a good supplier.

b. Standard-size plastic tissue-culture (Petri) dishes, ideally 60-mm diameter, but in any case large enough for watchglasses to be placed inside with forceps.

c. Disk filter papers cut to fit within dishes, and with central disk cutout for location and through-lighting of the watchglass.

d. Rings, at least 22 mm inner diameter and 3 mm depth, but with not more than 26 mm outer diameter. One face especially (the lower when set up) should form a plane and be smooth-surfaced. They are traditionally made of glass, but the skill to make them adequately is in short supply. We find rings cut from T.C.-grade stainless-steel tubing of 1.5 mm wall thickness, outer diameter 25 mm, and of 3 mm depth are ideal. One face and its edges should be polished, and the other with advantage left somewhat rougher to grip vitelline membranes.

8. Shelf air-incubator set to 38.5°C for ring culture and transient dish culture with oligos. We have not found CO2-buffered chick cultures advantageous, but some labs believe them to be. In that case, the TCM component for the culture itself should be different, although it should be pointed out that chick egg albumen itself is extremely alkaline and presumably matched to the embryo's requirements in this way.

9. Straight-sided, screw-capped plastic bottles of 5 mL for roller culture (13) (Sterilin, Elkay, Shrewsbury MA).

10. A roller-tube incubator, set so that the 5-mL bottles themselves rotate at 45 rpm. A cabinet fan-heated to a controlled 38.5°C houses a set of rollers with long axes inclined 10-15° off horizontal, humidification being unnecessary as tube caps are sealed. B.T.C. Engineering, Milton, Cambridge, UK, makes a compact bench-top model, the standard setting of whose roller apparatus at 30 rpm produces the required rotation of these tubes for chick culture.

11. Instruments for culture and manipulation of chick embryos:

a. Heavy blunt smooth forceps with ridged tips, for cracking eggshells, pulling albumen off yolk without tearing vitelline membranes and lifting watchglasses and rings (Subheading 3.1., Part 1).

b. Fine forceps (in UK no. 5 jeweler's) with close-fitting tips ground only slightly blunter than as from manufacturer, for gripping by their peripheries and moving individual blastoderms while off their vitelline membranes.

c. Two pairs of forceps as in item b, but with tips ground to be accurately fitting but relatively blunt and smooth, for manipulating vitelline membrane (Subheading 3.1., Part 2 and Subheading 3.4., steps 2 and 3).

Fig. 1. Ring-culture and off-membrane "oligo incubation": A young head-process-stage blastoderm (a typical stage for treatment) is seen in plan view at top. The middle diagram shows how such blastoderms are incubated, most often epiblast layer upward, in shallow layers of medium in plastic dishes (not to scale with blastoderm). The bottom sectional view is of such a blastoderm in the membrane-ring-watchglass assembly of New ring culture (12) over albumen medium, showing the germ-layer-inverted configuation and the adhesion of the epiblast periphery (normally, and ultimately again after successful replacement) to the membrane.

Fig. 1. Ring-culture and off-membrane "oligo incubation": A young head-process-stage blastoderm (a typical stage for treatment) is seen in plan view at top. The middle diagram shows how such blastoderms are incubated, most often epiblast layer upward, in shallow layers of medium in plastic dishes (not to scale with blastoderm). The bottom sectional view is of such a blastoderm in the membrane-ring-watchglass assembly of New ring culture (12) over albumen medium, showing the germ-layer-inverted configuation and the adhesion of the epiblast periphery (normally, and ultimately again after successful replacement) to the membrane.

d. Ideally, small curved-tipped forceps, with finely ridged tips, for lifting culture rings slightly after vitelline membranes have been wrapped and stretched

Fig. 2. Instruments for ring culture: In order from top to bottom, approximately real size, are shown a normal Pasteur pipet (the starting form), the special curved pipet for underfilling and draining cultures and settling blastoderms onto membranes, and the medium- and the wide-mouthed pipets for handling blastoderms. An appropriate shape for the smooth tungsten needle tip is shown in side view (left) and from above, to scale with the periphery of a blastoderm (right).

Fig. 2. Instruments for ring culture: In order from top to bottom, approximately real size, are shown a normal Pasteur pipet (the starting form), the special curved pipet for underfilling and draining cultures and settling blastoderms onto membranes, and the medium- and the wide-mouthed pipets for handling blastoderms. An appropriate shape for the smooth tungsten needle tip is shown in side view (left) and from above, to scale with the periphery of a blastoderm (right).

on them (Subheading 3.1., Part 2 and Subheading 3.3.). Failing these, the forceps of item c are used for this.

e. Medium straight-bladed scissors with pointed tips for cutting vitelline membrane rings off yolks (Subheading 3.1., Part 1).

f. Glass Pasteur pipets with carefully flame-smoothed mouth openings and sizes as shown in Fig. 2. These should have bulbs (teats) of the firmest rubber variety available. Within reason, the "tougher" the squeeze required on the bulb, the more delicate and controlled can be the sucking and blowing movements during set up of cultures and washing of blastoderms. The soft plastic "pastettes" (Alpha Labs. Ltd.), can be used as throw-away alternatives for all except the special curved pipet shown in the figure, which is essential for ring-culturing. In this the plane of the mouth should be sloped at some 45°, in order to be near-horizontal to bench surface when the pipet is held relaxed, with wrist on the bench.

g. An electrolytically smoothed tungsten needle with something like the tip shape and size, in relation to a blastoderm indicated in Fig. 2. A needle-mounter that is fat like a writing pen helps the hand stay relaxed for small controlled movements (Subheading 3.1., Part 2).

12. Last but strangely, not necessarily easiest to find are smooth-surfaced, circular or oval, flat-bottom glass or plastic dishes, of such a depth that a layer of saline just covering egg yolks comes within a centimeter or two of the rim (Subheading 3.1., Part 1). Black color at the dish bottom is advantageous, but a cleaned black microscope tile or something similar can be inserted for visualizing membranes and blastoderms.

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