Fig. 4

Fig. 4

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.