Site‐directed zebrafish transgenesis into single landing sites with the phiC31 integrase system

Abstract

Background Linear DNA-based and Tol2-mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long-term transgene stability unpredictable with variable expression. Targeted, site-directed transgene integration into pre-determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site-directed transgenesis. Results We report the implementation of the phiC31 integrase-mediated attP/attB recombination for site-directed zebrafish transgenics of attB-containing transgene vectors into single genomic attP landing sites. We generated Tol2-based single-insertion attP transgenic lines and established their performance in phiC31 integrase-catalyzed integration of an attB-containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue-specific transgene applications as well as long-term stability of phiC31-mediated transgenes. Conclusions Our results establish phiC31 integrase-controlled site-directed transgenesis into single, genomic attP sites as space-, time-, and labor-efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community.

Targeted transgene integration overcomes variability of position effects in zebrafish

“…Integration of the donor plasmid into a pseudo-att site is expected to yield YFP fluorescence in the absence of RFP fluorescence. We observed 100% concordance between red lens colour and YFP expression pattern, which indicates that such non-specific events are extremely rare, consistent with a recent publication by Mosimann and colleagues (Mosimann et al, 2013).…”

“…Integration of the donor plasmid into a pseudo-att site is expected to yield YFP fluorescence in the absence of RFP fluorescence. We observed 100% concordance between red lens colour and YFP expression pattern, which indicates that such non-specific events are extremely rare, consistent with a recent publication by Mosimann and colleagues (Mosimann et al, 2013).We chose insertion of full donor constructs as opposed to the previously published strategy of cassette exchange (Hu et al, 2011). Cassette replacement has the advantage that the vector backbone is not integrated, eliminating the potential impact of bacterial vector backbone on transgene expression.…”

“…Lister (Lister, 2010) demonstrated that PhiC31 integrase can facilitate intramolecular excision of a DNA sequence flanked by attP/attB sites in zebrafish (Lister, 2010;Lister, 2011). More recently, targeted integration of a transgene by PhiC31-mediated cassette exchange and insertion of circular plasmid were demonstrated in the genome of zebrafish (Hu et al, 2011;Mosimann et al, 2013).We have developed a simple phenotype-based strategy using eye colour change to monitor site-specific integration of a transgene into the zebrafish genome. We have reproducibly achieved an ~10% germline transmission rate with site-specific transgenesis using PhiC31.…”

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

“…To isolate genomic DNA from crispants or F1 mutants, single embryos of appropriate stages were incubated in 50 μl alkaline lysis buffer (25 mM NaOH, 0.2 mM disodium EDTA, pH 12.0) at 95°C for 30 min (Mosimann et al, 2013). The samples were then quenched on ice and neutralized with 5 µl of 1 M Tris-HCl pH 8.0).…”

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

“…Vectors with "insulator sequences" to protect the minimal promoter from position effects have been developed [14]. Additionally, both "knock-in" technology using homologous recombination [11], as well as efficient site-specific transgenesis using loxP sites [15][16][17][18], or the PhiC31 integrase [19,20], have been described to overcome this hurdle and the latter method used recently for testing enhancer activity in medaka.…”