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Fig. 4. The spatial pattern of x14-3-3~ expression. Whole-mount in situ hybridisation was carried out according to the protocol of Harland (1990).
The probe was synthesised using T7 (antisense) or T3 (sense) RNA polymerase in the presence of digoxigenin-UTP using recombinant pGEM-T
template containing the PCR ampification product from the 3'UTR of x14-3-3~ cDNA insert (see Fig. 1). Hybridisation was carried out for 16 h
at 52°C in 50% formamide, 2 x SSC, 1 mg/ml bacterial tRNA, 2% Boehringer-block (Boehringer), 0.1% Tween-20, 0.1% CHAPS and 0.1 mg/ml
heparin. Washes were carried out at 60°C in 2 x SSC, 0.3% CHAPS (3 x 20 rain each) and then in 0.2% SSC, 0.3% CHAPS (2 x 10 min each).
These represent high stringency conditions as demonstrated by our recent studies on the COUP-TF-A and COUP-TF-B genes, which show 94%
identity at the aa level (van der Wees et al., 1996). (A, B) Tailbud embryo (stage 36) probed with antisense and sense transcripts, respectively; (C,
D) early tadpole (stage 45) probed with antisense and sense transcripts, respectively, ba, branchial arches; n, notochord; ov, optic vesicles; s,
somites; sp, spinal cord. The result is representative of at least six independent embryos analysed. |
