Generation of attP/attB-recombinant zebrafish transgenics. A, B: cmlc2:EGFP-marked attP sites were introduced into the zebrafish genome by Tol2-mediated transgenesis. Presence of attP site allows for integration of attB-bearing transgene plasmid (shown ubi:EGFP as example) upon co-injection with phiC31 integrase mRNA into offspring from heterozygous cmlc2:EGFP-positive attP landing site carriers crossed out to wt; asterisks indicate cmlc2:EGFP-positive hearts. C: Injection of attB_ubi:EGFP together with phiC31 integrase mRNA causes mosaic fluorophore expression in the injected F0 larvae (top panel) and adult (bottom panel) of representative attP landing site lines, as detected by fluorescence microscopy; asterisk indicates cmlc2:EGFP-positive hearts. D: Agarose gel analysis of attL/attR-specific PCR on genomic DNA of 8 individual cmlc2:EGFP-positive, ubi:EGFP-mosaic embryos (48 hpf); arrows in the schematic indicate primer positioning, arrowheads highlight PCR product sizes. E: Sequencing of the verified precise attB plasmid integration into the genomic attP site. Blue boxes indicate the TTG crossover site. Sequencing of amplicons 1 through 8 from attL- and attR-specific amplicons verified precise attB plasmid integration into the genomic attP site by comparison to reference sequences (blue row). Representative sequencing graphs demonstrate sequencing results (bottom panels).

Germline integration and transgene transmission. A: Theoretical experimental outcome. Upon successful attP/attB recombination in the germline, outcrossing of attP, cmlc2:EGFP- and attB_ubi:EGFP-positive F0 adults to wt results in 50% wt and 50% cmlc2:EGFP-positive landing site carriers in the F1, with a fraction of additionally ubi:EGFP-positive embryos. B: Observed experimental outcome as outlined in A. Small inset shows through-light overlay to reveal the GFP-negative animal. C: Obtained transmission rates, ranging from 25% for attP^2A to 40% for attP^2B and 36% for attP^3B, compared to 2% background transmission in the wt control. D: In individual double-positive F1 embryos, faithful attP/attB recombination was tested by PCR with primers indicated as arrows and subsequent agarose gel electrophoresis; the gel depicts PCR results from 10 individual attP2B/attB_ubi:EGFP embryos, including positive and negative control lanes. E: EGFP fluorescence microscopy of 48-hpf embryos showing stable attP/attB transgenic F1 with comparable attB_ubi:EGFP-expression in each clutch as well as across clutches from two different founders (founder A and B), indicative of a single integration. F: Southern blot analysis of attP^2B with recombined ubi:EGFP probed for EGFP after EcoRV digest. Minus lanes depict uninjected, native attP^2B; plus lanes are ubi:EGFP-positive F1 zebrafish; numbers mark independent founders; the X marks untargeted GPF debris fragment, while the last two lanes show non-targeted concatemeric (c.) insertions from unspecific (unsp.) founders as reference. The ethidium bromide fluorescence shows the gel in F before transfer; the asterisk marks sample “Founder 2, fish 1” with less DNA input relative to the other lanes; M indicates the DNA marker lane. G: Southern blot analysis attP^2B with recombined ubi:EGFP probed for the genomic locus (digested with BglII); the white arrowhead indicates the native attP site band, the black arrowhead marks the size shift upon ubi:EGFP integration; also note the longer endogenous locus band (asterisks) as probed zebrafish were heterozygous for the transgenes, and high background due to repetitive elements in the probed genomic locus (see also Experimental Procedures section for details). H: Schematic of attP^2B genomic locus before and after ubi:EGFP integration, the distribution of the EcoRV and BglII restriction sites, and the locus-specific Southern probe. Green arrows indicate EGFP coding sequences and their respective orientation, the yellow bar shows the integrated ubi:EGFP vector.

Non-targeted integration detection. A: Schematic depicting how non-targeted, phiC-31 integrase-independent background insertions of the attB transgene plasmid can be recognized. Such events result in attP, cmlc2:EGFP-negative but attB_ubi:EGFP-positive embryos in the F1, referred to as non-targeted background integrations. B: Example of a recovered clutch with non-targeted integrations. All recovered events of this class stemmed from transgene vector breakage independent of phiC31 and outside of the attB cassette. See text for details; asterisks indicate cmlc2:EGFP-positive hearts.

Characterization of individual functional attP transgenic landing site lines. A: Example for enhancer trapping of an attP site transgene. Line attP^3B shows prominent EGFP expression in spinal cord neurons and the lateral line, indicating integration of the attP landing site in the proximity of a neuronal enhancer. Arrowheads indicate EGFP-misexpressing neurons, white dotted box marks area of close up. B, C: Flow cytometry analysis for EGFP expression in whole kidney marrow (WKM) of adult F1 attP/attB_ubi:EGFP transgenics to assess stable ubi:EGFP expression in the hematopoietic lineages. Despite strong overall EGFP expression (inset), line attP^2A shows no hematopoietic cells with EGFP (B), indicating transgene silencing in adult blood lineages. In contrast, line attP^2B (C) shows faithful EGFP expression in all adult hematopoietic populations. Representative flow cytometry analysis of individual animals is shown, separated by forward scatter (FSC) and side scatter (SSC); dotted black line indicates EGFP results for uninjected attP, cmlc2:EGFP control. D,E: Integrations shown as based on Ensembl Genome Browser; Zv9 assembly, modified to show transgene orientation. The genomic integration locus of individual attP sites was identified by TAIL-PCR using arbitrary degenerated primers binding in the genomic region adjacent to the attP insert together with nested Tol2-specific primers reading out of the transgene cassette (see Experimental Procedures section for details). D: attP^2AB maps to the first intron of the annotated gene PALM3 on chromosome 6, causing no obvious aberrant phenotype but resulting in silenced hematopoietic transgene expression. E: attP^2B maps to an approximately 650-kb-spanning intergenic region on chromosome 11, suggestive of a neutral insertion locus.

Tissue-specific promoter transgenes created using phiC31 integrase. A,B: pDestattB/CY, containing α-crystallin:Venus (CY, yellow fluorescence) as transgenesis marker (schematic), conveys eye lens-specific transgene expression when stably integrated into attP landing sites; the agarose gel illustrates positive (+) F1 transmission as detected by PCR from 2 representative independent F1 founders (A and B) including negative control siblings (), primers for attL indicated in schematic. Asterisk in B indicates EGFP expression from cmlc2:EGFP. C,D: Transgenics with an attB_mitfa^rescue transgene in mitfa-mutant nacre containing the attP^2A site; (C) schematic of the final transgene integration and initial adult phenotype without melanocytes in uninjected control (ctrl), compared to (D) adult transgenic with stable F1 attP^2A/attB_mitfa^rescue integration (inset shows skin close-up).