Kawakami K .

R01 GM069382 NIGMS NIH HHS

	Figure 1. The structures of the Tol2 transposable element and the minimal Tol2 vector. At the top of the illustration is the 4,682 base pair (bp), full-length Tol2. RNA transcribed from Tol2 that encodes a transposase protein [3] is shown by lines (exons) and dotted lines (introns). Black boxes and gray boxes represent coding regions and untranslated regions, respectively. Black arrowheads in boxes at both ends indicate 12 bp terminal inverted repeats (TIRs). The lower portion of the figure shows the minimal Tol2 vector with the green fluorescent protein (GFP) expression cassette. The minimal Tol2 vector contains 200 and 150 bp of DNA from the left and right ends, which include TIRs (black arrows) and subterminal regions (open boxes) [5]. The transposon vector can carry a DNA fragment, for example the GFP expression cassette in this figure, between these sequences.
	Figure 2. Transgenesis in zebrafish. The synthetic transposase mRNA and a transposon donor plasmid containing a Tol2 construct with a promoter and the gene encoding green fluorescent protein (GFP) are co-injected into zebrafish fertilized eggs. The Tol2 construct is excised from the donor plasmid [2] and integrated into the genome. Tol2 insertions created in germ cells are transmitted to the F1 generation. Germ cells of the injected fish are mosaic, and, by crossing the injected fish (founder) with wild-type fish, nontransgenic fish and transgenic fish heterozygous for the Tol2 insertion are obtained [4]. In this figure, the promoter is tentatively defined as a spinal cord specific enhancer/promoter and the spinal cord of the embryo is depicted in green.
	Figure 3. Transient expression assay in zebrafish. The synthetic transposase mRNA and a transposon donor plasmid containing a Tol2 construct with the tentative spinal cord specific enhancer/promoter (Figure 2) and the gene encoding green fluorescent protein (GFP) are co-injected into zebrafish fertilized eggs. The Tol2 construct is excised from the donor plasmid and integrated into the genome of somatic cells. The activity of the enhancer can be visualized in the injected embryo, because GFP is expressed predominantly in a region where the enhancer/promoter should activate transcription [22].
	Figure 4. Transgenesis in Xenopus tropicalis. The synthetic transposase mRNA and a transposon donor plasmid containing a Tol2 construct with a ubiquitous promoter and the gene encoding green fluorescent protein (GFP) are co-injected into X. tropicalis fertilized eggs. The Tol2 construct is excised from the donor plasmid [25] and integrated into the genome. In the study conducted by Hamlet and coworkers [26], injected GFP-positive tadpoles were raised to adulthood. Tol2 insertions created in germ cells are transmitted to the F1 generation. Germ cells of the injected frogs are mosaic, and, by crossing theinjected frog (founder) with wild-type frog, nontransgenic tadpoles and transgenic tadpoles heterozygous for the Tol2 insertion are obtained [26].
	Figure 5. Transient expression assay in chicken. A plasmid DNA containing the transposase cDNA under the control of a ubiquitous promoter (CAGGS) and a transposon-donor plasmid DNA containing a Tol2 construct with the CAGGS promoter and the gene encoding green fluorescent protein (GFP) are introduced into the somite of a chicken embryo by electroporation. The Tol2 construct is excised from the plasmid and integrated in the genome. GFP expression in the somites persists until late developmental stages [28].
	Figure 6. Chromosomal integration in mammalian culture cells. A plasmid DNA containing the transposase cDNA under the control of a ubiquitous promoter (CAGGS) and a transposon-donor plasmid DNA containing a Tol2 construct with a ubiquitous promoter (the phosphoglycerate kinase [PGK] promoter used by Kawakami and Noda [33]) and the neomycin-resistance gene are introduced into mammalian culture cells by electroporation. The Tol2 construct is excised from the plasmid and integrated into the genome. Chromosomal integration of the Tol2 construct is enhanced, depending on transposase activity [33].

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