Stable Tol2-BAC transgenesis in zebrafish and mice. (a, b) Side views of double transgenic larvae (F1-18 and F1-43) carrying Tol2-Frevx1:Gal4-BAC (Tol2-BAC) and UAS:GFP that expressed GFP in spinal cord neurons at 2 dpf. (c) The insertion in F1-18 was located ∼50 kb upstream of cng3. (d) The insertion in F1-43 was located in intron 7 of ntt4. 8 bp target site duplications are shown. (e) The structure of Tol2-BAC. Tol2 cis-sequences are shown by red triangles. Black bars indicate exons of hoxa and evx1 genes. A purple box indicates Gal4FF. The pBeloBAC11 vector is shown in gray and a black arrow within it is chloramphenicol. Positions of PCR fragments amplified (g, j) are shown as red lines with numbers. Positions of the BamHI, NheI and EcoRV sites used for Southern blot are shown. (f) Southern blot analysis of transgenic fish (F1-18 and F1-43) by using a ³²P-labeled Gal4FF probe. Single bands (arrowheads) were detected in DNA digested with EcoRV (∼19-22 kb) and NheI (~21 kb). (g) Electrophoresis of PCR products amplified from transgenic fish. (h) PCR genotyping of F1 progeny from No.1 and No.3 founders mice with Gal4FF primers. (i) Southern blot analysis of F1 transgenic mice by using a ³²P-labeled Gal4FF (left and middle panels) or Chloramphenicol (rightmost panel) probe. Single bands (arrowheads) were detected in DNA digested with EcoRV (∼23 kb) or BamHI (∼20 kb). (j) Electrophoresis of PCR products amplified from transgenic mice. M, 1 kb ladder. (k) The F1-3A insertion was located near Pomc. (l) The F1-3B insertion was located in AAHY01196472.1.

Tol2-mediated BAC excision in early zebrafish embryos. Transient in vivo assay of Tol2-mediated BAC excision in zebrafish embryos, including DNA microinjection procedure and demonstration of iTol2 cassette excision by PCR.

Estimation of Tol2-BAC copy number in transgenic zebrafish and mice. Semi-quantitative PCR on BAC transgenic zebrafish and mouse genomic DNAs. PCR gel band intensities generated from transgenic DNAs were compared to those of control BAC DNA standards to produce an estimate of copy number.

Tol2-mediated excision of a 120 kb BAC clone in zebrafish embryos. Schematic of a 120 kb zebrafish BAC clone containing an iTol2 cassette and detection of its excision in vivo by PCR.