Journal: bioRxiv

Article Title: Library transgenesis in zebrafish through delayed site-specific mosaic integration for in vivo pooled screening of transgenes

doi: 10.64898/2026.01.30.702415

Figure Lengend Snippet: (A) A single-copy genomic AttP landing site (orange) in the pIGLET (‘phiC31 Integrase Genomic Loci Engineered for Transgenesis’) line , with an exogenously introduced plasmid containing an AttB site (blue) and a transgene cassette (green). Once the PhiC31 integrase enzyme (red) is introduced, it catalyzes recombination between the AttP and AttB, leading to single-copy genomic integration of the plasmid. (B) Schematic of the overall procedure of delayed site-specific library transgenesis. (i) The 1-cell embryo is injected with a mixture of plasmids (the transgene library, drawn as circles with blue, magenta and green rectangles) and mRNA encoding for the PhiC31 integrase (red). (ii) During early development, the library passively spreads in the embryo as episomal plasmids together with the PhiC31 mRNA/protein as the cells divide. (iii) After an initial stage of development, the PhiC31 becomes active and integrates a single randomly-selected plasmid from the library in each cell. (iv) This produces a mosaic animal in which different cells express different library members, and only one library member in each cell.

Article Snippet: The PhiC31 plasmid (Addgene #68310) was used to produce purified PhiC31 mRNA using the mMESSAGE mMACHINETM T7 Transcription Kit (Thermo Fisher, AM1344) with lithium chloride purification.

Techniques: Plasmid Preparation, Injection

Journal: bioRxiv

Article Title: Library transgenesis in zebrafish through delayed site-specific mosaic integration for in vivo pooled screening of transgenes

doi: 10.64898/2026.01.30.702415

Figure Lengend Snippet: pIGLET heterozygous larvae display ∼99% mutually exclusive mosaic expression of a single library member per neuron. ( A ) Illustration of the experiment: pIGLET heterozygous embryos containing a single AttP site in chromosome 14 or 24 were injected with PhiC31 mRNA and a 50:50 mixture of plasmids containing an AttB site (blue) and constructs for neuronal expression of mScarlet (magenta) or GFP (green), fused to a CAAX tag for membrane targeting. 3 or 5 days later, the larvae were imaged. ( B-E ) Representative images of a mosaic 5 dpf pIGLET24b heterozygous animal (pIGLET24b;HuC::Gal4;nacre;RH1::DsRed) following library transgenesis, showing the forebrain and midbrain (B), midbrain and hindbrain (C), posterior hindbrain and spinal cord (D) and spinal cord (E). Max-projection images are shown with skin autofluorescence removed to aid visualization. ( F ) One plane of the fluorescent channels overlaid on a brightfield image of a mosaic 5 dpf pIGLET14a heterozygous larva (pIGLET14a;HuC::Gal4;nacre;RH1::DsRed, with prominent expression of the red eye marker). ( G ) Zoomed-in image of the section marked with a cyan box in the hindbrain in (C), showing a neuron co-expressing GFP and mScarlet. ( H ) Quantification of the ratio of neurons expressing both GFP and mScarlet (double-positives), out of all transduced neurons, in 8 mosaic larvae. The animals for which the whole hindbrain was quantified are marked in orange. The 3 dpf larva is marked in blue. The rest (marked in gray) are 5 dpf larvae for which 1-4 random FOVs were quantified. All the raw numbers are available in . ( I-L ) As in B-E, but for a 3 dpf larva. Scale bar: 50 μm.

Article Snippet: The PhiC31 plasmid (Addgene #68310) was used to produce purified PhiC31 mRNA using the mMESSAGE mMACHINETM T7 Transcription Kit (Thermo Fisher, AM1344) with lithium chloride purification.

Techniques: Expressing, Injection, Construct, Membrane, Marker

Journal: bioRxiv

Article Title: Library transgenesis in zebrafish through delayed site-specific mosaic integration for in vivo pooled screening of transgenes

doi: 10.64898/2026.01.30.702415

Figure Lengend Snippet: ( A ) Construct design for the barcoded GFP-CAAX library plasmids. Each plasmid includes an AttB sequence (light blue), HS4 insulator element (medium gray) embedded with a unique 15-nt random sequence barcode (15xN, purple), a 4xnrUAS minimal promoter for tissue-specific Gal4 transcription activation (light gray), GFP-CAAX (green arrow) and SV40 polyA (dark gray). ( B ) Scheme illustrating the experiment-a high-complexity library of barcoded GFP-CAAX plasmids, each containing a different 15xN barcode (dark blue, magenta and yellow rectangles on the plasmids), was injected into 1-cell embryos of heterozygous pIGLET24 zebrafish containing a genomic AttP site on chromosome 24. The library was co-injected together with mRNA encoding for the PhiC31 integrase (red). 5 days later, the larvae were imaged to confirm GFP expression in neurons, and then selected for extraction of genomic DNA from their entire bodies (n=12 larvae). ( C ) the genomic extracts were used as templates for 10-cycle PCR amplification of the barcodes from all genomically integrated plasmids, using primers that specifically target the integration junction (shown in red arrows). The resulting 650-bp amplicon library is then re-amplified with different, internal primers (shown in blue arrows), to attach overhangs for Illumina next generation sequencing (blue) and add a 5-nt sample-specific multiplexing barcode (pink) for pooled sequencing. ( D ) Number of unique high-confidence barcodes identified in each fish, after barcode collapsing and filtering. ( E ) Histogram of barcode abundance for the injected source library and for the barcodes recovered from the fish-integrated plasmids. The injected library displays a narrow distribution, indicating high complexity (many rare barcodes appearing at similar low frequency) while the fish-recovered barcodes display a broader long-tailed distribution (some barcodes appearing much more than others), consistent with intra-fish clonal expansion of the integrated transgenes. The per-fish barcode abundance histograms are available in . RPM=reads per millions (read counts normalized to the total number of reads sequenced for each sample). ( F ) Nucleotide composition for each position in the injected library and in the integrated barcodes shows high sequence diversity and no sequence bias for integration. Per-fish barcode sequence compositions are available in .

Article Snippet: The PhiC31 plasmid (Addgene #68310) was used to produce purified PhiC31 mRNA using the mMESSAGE mMACHINETM T7 Transcription Kit (Thermo Fisher, AM1344) with lithium chloride purification.

Techniques: Construct, Plasmid Preparation, Sequencing, Activation Assay, Injection, Expressing, Extraction, Amplification, Next-Generation Sequencing, Multiplexing