A set of transgenic insertion stocks derived by TE mobilization using the Dhyd\Minos-based construct Mi{MIC}; created and vetted by the Gene Disruption Project (GDP). The Mi{MIC} construct carries the mini-yellow marker y+mDint2 and Avic\GFP (a fluorescent marker) associated with a mutagenic gene-trap cassette. Two phiC31\attP sites are located immediately within the Dhyd\Minos termini, flanking the EGFP and yellow markers, allowing replacement with DNA of interest using Recombination Mediated Cassette Exchange (RMCE) methodology.
Additional lines were created and assessed; in total, the collection comprises 7434 insertions in 7400 lines associated with 4367 genes; 34 lines contain two insertions each. Insertions within introns flanked by coding exons were characterized further and described as:
Gold set: 1732 insertions in constitutive coding introns that permit tagging of all annotated protein isoforms.
Silver set: 814 insertions in alternative coding introns that permit tagging of more than 50% of annotated protein isoforms.
Bronze set: 328 insertions in alternative coding that permit tagging of less than 50% of annotated protein isoforms.
Initial experiments allowed recovered of new insertions on the autosomes. Mobilization occurred in animals carrying one of 5 Mi{MIC} insertions on the X chromosome and P{hsILMiT}, a source of Dhyd\Minos transposase, on a second chromosome balancer (CyO-MiT2.4). In subsequent experiments, one of 3 insertions of Mi{MIC} on a third chromsome balancer (TM3-Sb) was used, allowing recovery of new insertions on the X chromosome as well as the autosomes. 4,464 single-insertion Mi{MIC} lines were created; new lines continued be generated.
Mi{MIC} lines up to MI04464 are described in Venken et al., 2011 (FBrf0216331): subsequent lines are described in Nagarkar-Jaiswal et al., 2015 (FBrf0228008).
Flanking genomic sequences were obtained by sequencing of inverse PCR products, and were mapped by alignment to the genomic sequence; unique insertion sites were determined for 3,633 insertions. Analyses of insertions within UTR and coding introns demonstrated the usefulness of the ability to insert different gene-trap and protein-trap cassettes by RMCE, allowing visualization of gene expression patterns and subcellular protein distributions with high resolution.
