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Fig. 1. A two-step cloning procedure to ameliorate DNA recombination during construction of plasmids bearing two dual flanking insulators with inverted orientation. See the text (Materials and methods) for details.
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Fig. 2. Frequency of different expression patterns in stages (st.) 43–46 tadpoles, injected at the 2-cell stage with plasmids containing glGFP, an attB site, and two flanking dual insulators (as in Fig. 1), driven by three separate promoters [neuronal class II β tubulin (NβT), neurofilament medium (NF-M), and cardiac actin (CarA)]. Total numbers of surviving injected animals that were scored for each group are as indicated (N). Pictures show dorsal or dorsolateral views of live tadpoles, taken with a Nikon DSRi1 (color) camera through a fluorescence stereo microscope. Percentages of tadpoles presenting different expression patterns are indicated below the pictures of corresponding patterns. (Rows A1–B4) Reporter protein expression driven by the two neuronal promoters (NβT, NF-M) is categorized into 1) strong promoter-typic expression located in the nervous system (neural), 2) promoter-typic expression in the nervous system with weak expression outside the nervous system, predominantly in tail muscle (neural+weak muscle), 3) non-promoter typic, mosaic expression, largely in muscle or epidermal cells, with or without some expression in the nervous system (ectopic, shown here are examples of no neuronal expression), and 4) undetectable expression (no expression). (Row C1–C4) Reporter protein expression driven by the CarA promoter is categorized into 1) consistent, saturated promoter typic expression throughout somitic muscle fibers (high penetrance), 2) consistent promoter typic expression in somitic muscle with lower intensity than the first category (medium penetrance), 3) sporadic expression, which, although restricted to muscle cells, varies in intensity among muscle fibers, leaving gaps that lack expression entirely (low penetrance), and 4) undetectable expression (no expression). Arrowheads and arrow point to brain, spinal cord, muscle cells, tail muscle, and gap without expression, as indicated. Dotted lines in C1–C3 outline tail regions. The gut, which is autofluorescent, is also labeled on all pictures.
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Fig. 3. Detailed examples of robust, highly promoter-typic reporter protein expression (category 1 of Table 1) in st. 43–46 tadpoles injected with constructs bearing different promoters (columns B1–4, NβT; C1–4, NF-M; D1–4, CarA). (Row A1–D1) Views through a fluorescence stereo microscope of live tadpoles. Dotted lines outline the brain and spinal cord (A1–C1); arrowheads point to cells and anatomical structures as labeled; the apparent label in gut is autofluorescence (all panels). Whereas the lower power images were photographed with a DFC 340 FX monochrome camera, that of the inset, which depicts a close-up of the ventral region of a tadpole surrounding the heart, was photographed with a DSRi1 color camera (brightness and contrast were dynamically adjusted in this image to aid visualization of the dimmer, emerald green GFP expression against the brighter, yellow autofluorescence of the gut). Subsequent images are transverse sections of the eye (row A2–D2), hindbrain (row A3–D3), and spinal cord (outlined by dotted line; row A4–D4), immunostained for GFP. Arrowheads point to retina, lens, neuron, and muscle cell as labeled directly on the images. (Column A1–4) GFP expression is absent in uninjected controls. (Columns B1–4, C1–4) Robust GFP expression is seen in neurons of tadpoles injected with constructs bearing the NβT and NF-M promoters, respectively. Both neuronal cell bodies and axons are seen on the injected side (left side of sections in B3–C4), but only decussated axons are visible on the uninjected side (right side of sections in B3–C4). (Column D1–4) GFP expression is seen in muscle (somites: D1,3,4; jaw musculature, D1 inset), and heart (D1, inset) of tadpoles injected with the construct containing the CarA promoter. Scale bars, 50 μm.
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Fig. 4. Characterization of promoter-typic reporter protein expression at early developmental stages in live tadpoles injected with constructs bearing different promoters, corresponding to the first categories in Table 2 (columns A1–4, uninjected control; B1–4, NβT; C1–4, NF-M; D1–4, CarA). (Row A1–D1) st. 18–20; (row A2–D2) st. 22–24; (row A3–D3) st. 32–33/34; (row A4–D4) st. 39–40. Labeled arrowheads point to anatomical regions of emerging promoter-typic expression. Scale bars in rows A1–D1/A2–D2/A3–D3 and in row A4–D4 are 500 μm and 1 mm, respectively.
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Fig. 5. Persistent, promoter-typic reporter protein expression required injection of a plasmid containing an attB site plus two dual insulators. Plasmid maps for the corresponding groups are shown above each column, with the presence or absence of an attB site and one or two dual insulators, as indicated by the symbols (+ or −, respectively). Total numbers of surviving animals scored for each group at st. 43–46 are indicated (N). Table at the bottom shows percentages of tadpoles bearing different expression patterns as characterized in Table 1 and Table 2. (A–E) Dorsolateral views of the heads and trunks of live st. 43–46 tadpoles, taken with a Leica DFC 340 FX monochrome camera through a fluorescence stereo microscope, illustrate representative examples from the category with the highest frequency of expression pattern from each group. Most tadpoles injected with plasmid lacking all three elements (A) exhibited only very weak, barely detectable expression that was mostly ectopic. All three groups injected with plasmids containing the attB site yielded robust expression in the CNS (B,C,E), but among these, those lacking either one (C) or two (B) dual insulators exhibited significantly more ectopic expression in muscle and epidermis (arrows) than did those possessing all three elements (E). Groups injected with plasmid lacking the attB element but having both insulators (D) exhibited only very weak to undetectable expression at these stages. Dotted lines outline the brain and spinal cord; autofluorescence from the gut is labeled on all panels.
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Fig. 6. Expression of coinjected plasmids differing only in their reporter sequences overlapped extensively. The GFP-expressing plasmid bearing an attB site plus two dual insulators (top left) was coinjected (75 pg each) with its DsRed2-expressing counterpart (top right). (A–D׳) Extensive overlap of labeled cells is seen in transverse histological sections of the same animal, immunostained for GFP (green, A–D) and DsRed2 (magenta, A׳–D׳). (A,A׳) hindbrain; (B,B׳) spinal cord; (C,C׳) eye; (D,D׳) a single neuron from hindbrain. Percentages of overlapping cells are presented on the right for hindbrain, spinal cord, and eye on the injected side. For each anatomical structure, percentages represent averages (±SD) over 3 animals, with 3 different sections taken from each animal. Scale bars in A–C′, 50 μm; D,D′, 20 μm.
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Fig. 7. Truncation of the 1.5 kb NF-M promoter to 0.4 kb increased expression outside the nervous system. (A) Dorsal view of a representative example of 1.5 kb NF-M:DsRed2kb expression in a tadpole at st. 43–46 (map of the injected plasmid is shown on the right). Expression is seen predominantly in the brain and spinal cord (arrowhead) of the injected side; autofluorescence in the gut is labeled. (B,C) Injection at the 2-cell stage of a new DsRed2-expressing construct bearing a truncated, 0.4 kb NF-M promoter (75 pg, plasmid map on the right) yielded two expression patterns with similar frequency in st. 43–46 tadpoles [54% (B) and 46% (C) of 106 scored animals]. Both patterns exhibited higher levels of expression overall and more expression in muscle and surrounding tissues than did the longer, 1.5 kb NF-M promoter. Arrowhead in B labels expression in muscle, at approximately the same rostrocaudal level as the arrowhead pointing to the spinal cord in A. (D–F׳) This new construct bearing the 0.4 kb promoter (plasmid map shown again in the third row, now designated as “Test”; 75 pg injected) was then coinjected with the GFP-expressing reference plasmid, which used the longer, 1.5 kb promoter (plasmid map shown on the bottom row, designated as “Reference”; 75 pg injected). Animals that expressed GFP predominantly in the nervous system (categories 1 and 2) were selected for analysis and protein expression was assayed by immunostaining transverse histological sections. Representative sections from animals that gave expression similar to that shown in C, immunostained for DsRed2 (D–F) and GFP (D׳–F׳), are shown. GFP expression driven by the reference plasmid in these animals appeared primarily in the nervous system [e.g., hindbrain (D׳) and spinal cord (E׳; staining seen in adjacent somites is predominantly in peripheral axons)], but not in gut (F׳), whereas DsRed2 expression from the test plasmid appeared not only in neural tissues [e.g., neurons in hindbrain (D) and spinal cord (E)], but also in non-neural tissues [e.g., somitic muscle cells (arrowheads, D,E) and gut (arrowhead, F)]. Scale bars, 50 μm.
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