Figure 1

A scheme for Tol2 -mediated BAC transgenesis. (a) Structures of Tol2 and iTol2 cassette. Tol2 encodes a single transposase mRNA (solid and dotted lines). The cis-sequences required for transposition, 200 bp from the left (L200) and 150 bp from the right (R150) are shown by red triangles. In the iTol2 cassette, these are inverted outwards. (b) The cassette was inserted into a Fugu evx1 BAC tagged with Gal4FF (purple box) that included part of the hoxAa cluster. The gray box indicates the pBeloBAC11 vector. The Tol2-BAC was co-injected with the transposase mRNA into zebrafish or mouse eggs. The Tol2-BAC portion was excised in vivo and integrates as a single copy into a single genomic locus (asterisk). (c) 50 ng/μl Tol2-BAC and 25 ng/μl transposase mRNA were co-injected into one-cell stage zebrafish embryos, or 14.5 ng/μl DNA and 10 ng/μl transposase mRNA into mouse oocytes (B6C3F1). A single needle was used to inject the DNA/RNA mixture into either pronucleus, cytoplasm or both of mouse oocytes.