Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes - PubMed (original) (raw)

Fig. 5

Structure of MGH mRNAs in liver of transgenic mice. a, Northern blot analysis. RNAs were resuspended 10 mM NaH2PO4, _p_H 7.4/50% formamide/2.2 M formaldehyde, heated at 65 °C for 5 min, and subjected to electrophoresis on a slab gel composed of 1.5% agarose in 10 mM NaH2PO4, _p_H 7.4/0.55 mM EDTA/1.1 M formaldehyde. The running buffer was 10 mM NaH2PO4, _p_H 7.4/0.5 M formaldehyde. The gel was stained with acridine orange to identify rRNA markers, photographed, incubated for 90 min with 50 mM NaOH, and then neutralized with two washes of 0.2 M NaOAc, _p_H 4.3. The RNA was then transferred to diazotized paper and prehybridized as described elsewhere. Lane 1, total liver RNA (15 μg) from a control littermate; lane 2, total liver RNA (15 μg) from MGH-21; 3, total mouse pituitary RNA; 4, 40 ng of rat pituitary mRNA, poly(A)+. Lanes 1 and 2 exposed for 48 h; lanes 3 and 4 exposed for 5 h. b, Single-strand-specific nuclease protection assay. 20 μg of RNA were hybridized at 47 °C for 5 h in 40 μl of a solution containing 40 mM PIPES _p_H 6.4, 0.4 M NaCl, 80% formamide and 50,000 c.p.m. gel-purified 221-bp _Sst_I–_Xho_I fragment of pMGH end-labelled at the Xho_I site with 32P (see Fig. 1_c). The samples were then diluted with 0.3 ml of 280 mM NaCl, 30 mM NaOAc _p_H 4.4, 4.5 mM ZnSO4, 20 μg ml−1 salmon sperm DNA and 150 units of mung bean single-strand-specific nuclease (Collaborative Research) and incubated at 47 °C for 1 h. Samples were ethanol-precipitated, resuspended in 90% formamide containing bromophenol blue and Xylene Cyanol FF, loaded on to an 8% acrylamide–urea sequencing gel, electrophoresed for 1.5 h at 2,000 V, dried and autoradiographed for 7 days at −70 °C with an intensifying screen. Lanes 1 and 2, sequencing ladder used for size standards; 3, MGH-3 RNA; 4, control liver RNA; 5, mouse pituitary RNA; 6, MGH-21 RNA.