Fig. 1.

Schematic summary for site-specific ϕC31 integrase-mediated transgenesis via pronuclear injection in mice. A single or three tandem attP sites were knocked into the Hipp11 (H11) or Rosa26 loci via homologous recombination in ES cells (Left; for details, see Fig. S1). Mice homozygous for one of the modified loci (H11 is shown as an example) served as embryo donors. A mix of DNA and in vitro transcribed ϕC31 mRNA was injected into a single pronucleus of each zygote. The integration of plasmid bacterial backbone (BB) that decreases the transgene expression was avoided either by injecting a minicircle DNA with a single attB site (top branch; in this case, ϕC31 catalyzes a typical integration reaction), or by injecting plasmid DNA where the gene of interest (e.g., GFP) was flanked by two attB sites (bottom branch; in this case, ϕC31 catalyzes a recombinase-mediated cassette exchange reaction). Right, Middle: Green fluorescence of a representative transgenic F0 embryo and the corresponding PCR results that indicate site-specific insertion. The red numbers for the PCR results correspond to the numbers on the H11P3-pCA-GFP transgene scheme below; the same PCR tests can be used for the transgene above. The particular embryo shown was obtained by cassette exchange. Inset: Bright-field image of the same embryo.