With Phosphorothioate Deoxyoligonucleotides

Blastoderms/embryos younger than stage 4, the full-length streak, tend to show poor subsequent development, such as small axial structures or abortive posterior segmentation, if incubated in the way to be described for more than 1-1.5 h. Older ones up to stage 9 (6-7 somites—beyond which blastoderms are difficult to replace stably into ring culture) are amenable to up to 3+ h of treatment. In any case, the conditions of the static off-membrane culture itself, rather than toxicity of oligos, appear to be the physiological limitation unless oligo concentrations are above 80 pM.

1. Remove subgerminal yolk from any stored blastoderms that left the membrane by themselves. Then gently wash all blastoderms by passing in and out of a medium-mouth pipet or pulling them around by the edges with smooth forceps, in a new dish of room temperature TCM:BSS, before careful transfer to the final 35-mm plastic Petri culture dish containing a measured quantity, such as 3 mL of the same medium. Such a dish can appropriately contain 12 streak-stage blastoderms, or fewer older ones, without overlap of the spread cell layers. The point of knowing the volume of the bath, from here on, is that with precise removals and additions of further components, the final concentrations of oligo DNA/ lipofection agents present can be known. With practice ("tough" pipet bulbs and relaxed supported wrists), blastoderms can be passed singly or in groups from the end of the blunt or medium pipet without contact with the surface, and with addition of only negligible volumes of medium to this "oligo bath". Concentration of the original egg albumen in the oligo bath should be <0.5% to avoid danger of phosphorothioate oligo precipitation. This is easy to achieve, but as a beginning check, mix 1:200 of the albumen medium into a dish of TCM:BSS to see the pronouncedly more purple color this produces. As a precaution against premature oligo degradation by exonucleases, we advise transition during this step to use of glass and metal instruments that are reserved solely for contact with oligo incubations, and can be water-washed, alcohol-treated, and flamed between uses.

2. The simple addition of oligos: Using automatic pipeters, such as Gilson's, the TCM:BSS in the dish is reduced to 0.8 mL, a layer just covering the spread blastoderms, meanwhile setting 0.7 mL aside by using it to take up the oligo from two to three freshly thawed aliquots in 0.5-mL Eppendorf tubes. Convenient aliquots of 12.5 pL of oligos at 2 mM in water will contribute 25 pM oligo/mL each to the final mixture. Thus, in the present case with final volume of 1.5 mL, two aliquots will give 34 pM and 3 aliquots, 50 pM treatment, with the option of studying the combined effects of different oligo sequences. The 0.7 mL is simply dripped on from successive fillings of a fine Gilson tip, taking care to cover all the embryos. With practice, the dripping can be of a size and height that visibly stretches and deforms the blastoderms' cellular surface without tearing it, which may help in penetration between cells, and so on, where a microlayer not subject to good bulk mixing may otherwise exist. This detail is probably only important for liposomes, however (step 3), which are supramolecular particles and not diffusing. Finally, blastoderms are pulled through the medium by their edges, or subirrigated by the fine pipet with medium already in the dish (no bubbles!), ensuring access to the side not covered by the dripping on. Incubate lidded dish for 1-3 h at 38.5°C, depending on blastoderm stage, swirling gently and respreading blastoderms at half-time. For crowded blastoderms, a small supplement of fresh medium at this time-point relieves acidification.

3. Addition of oligos with a lipofection procedure: In our hands, a procedure using Lipofectamine (Gibco-BRL), a recently available polycationic liposome-form-ing reagent (see, e.g., 15-17 for lipofection), gives appreciable if not dramatic potentiation of the sequence-specific effects per micromole of antisense DNA. Because the lipofection procedure involves the settling out of a fine suspension rather than presence of all the oligo in true solution, blastoderms should probably be placed with the surface uppermost that is believed most relevant for interference with the gene concerned. Otherwise, epiblast-up culture seems somewhat better physiologically, and we have in fact seen only modest evidence of the relevance of germ layer presentation. Lipofectamine is used at 0.5 |L (1.0 |g)/mL per micromole of oligo in the final lipofection medium, but this involves much less than the molar ratio of lipid to DNA believed optimal for liposome formation (see manufacturer's recommendations for transfections with "genetic" length DNA molecules). Chick blastoderms can be incubated in up to 25 | L/mL Lipofectamine for over an hour with normal long-term development, but absence of accompanying DNA makes the cells extremely sticky for plastic. We thus typically premix 1/15 of the oligo DNA complement for each incubation with the whole of the Lipofectamine for 20 min at room temperature, giving about the optimal DNA/lipid ratios (manufacturer's instructions) before addition to the blastoderms. This is immediately followed by the balance of the naked oligo DNA to give the planned total oligo micromolarity. There is evidence from cell-culture systems that access of the additional naked DNA into the relevant cell compartments (nucleus and cypoplasm) may be potentiated by the original lipofection (9). Incubation with such a liposome preparation alone, thus, including 2-3 ||M S-oligo DNA with acceptable Lipofectamine concentrations, does not give effects on whole chick embryos. We thus give below a typical treatment, avoiding DNA precipitation and giving optimal sequence-specific effects from 50 |M phosphorothioate oligos (15-mer), though with many S-oligo samples the simple addition of all the DNA to the lipid in a total volume of at least 0.5 mL, 20 min before presenting to the blastoderms, works as well;

a. Take up the oligo or oligos from aliquots as in step 2 above (e.g., 3 aliquots of 12.5 |L at 2 mM for a final 1.5 mL of 50 |M), in 0.3 mL total TCM:BSS, and take to one tube. Then place a further 0.2 mL of such medium into each of two small plastic tubes of materials recommended in the technical data sheet for Lipofectamine. Original Eppendorf tubes as well as some others appear to be suitable. To one tube add 25 |L (50 |g) Lipofectamine; to the other, 145 (i.e., 22 |L in total) of the solution of naked DNA oligo or mix of oligos. Mix each tube rapidly, then mix together in one of the tubes, but without foaming, and leave for 20 min at room temperature. This mixture is left at room temperature for 20 min for liposome formation, so the step is suitably done before the final assembly, washing, and sorting of off-membrane blastoderms into the treatment dishes (experimental and matched control group, and so on). The result should be an opalescent appearance in the tube, but not a turbid precipitate. Precipitation follows either contamination of the TCM:BSS with egg albumen, or allowing the local concentration of oligo to rise above 150 |M on mixing with the lipid.

b. Remove medium from each "oligo dish" containing its blastoderms, to leave the shallow layer of 0.8 mL, then drip the lipofection mixture (0.4 mL) onto the chosen surface of the blastoderms in the dish as for simple oligo treament (step 2). Immediately follow this with the (0.3 mL) balance of the naked oligo DNA in TCM:BSS medium, bringing the volume up to approx 1.5 mL.

c. Using flamed smooth fine forceps, "pull" the embryos about through the medium layer to mix the liposome-containing medium intimately into the cellular surface, and subirrigate with a fine pipet avoiding air bubbles. Leave them spread out epiblast- or hypoblast side uppermost (depending on desired target site for oligos). Incubate the lidded dish at 38.5°C for 1-3 h (2 h are our most frequent period with headfold stage embryos of 1-3 somites), possibly remixing gently and "feeding" (see step 2 above), once during the incubation period.

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