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Thibault, Stephen T; Singer, Matthew A; Miyazaki, Wesley Y; Milash, Brett; Dompe, Nicholas A; Singh, Carol M; Buchholz, Ross; Demsky, Madelyn; Fawcett, Robert; Francis-Lang, Helen L; Ryner, Lisa; Cheung, Lai Man; Chong, Angela; Erickson, Cathy; Fisher, William W; Greer, Kimberly; Hartouni, Stephanie R; Howie, Elizabeth; Jakkula, Lakshmi; Joo, Daniel; Killpack, Keith; Laufer, Alex; Mazzotta, Julie; Smith, Ronald D; Stevens, Lynn M; Stuber, Christiana; Tan, Lory R; Ventura, Richard; Woo, Alesa; Zakrajsek, Irena; Zhao, Lora; Chen, Feng; Swimmer, Candace; Kopczynski, Casey; Duyk, Geoffrey; Winberg, Margaret L; Margolis, Jonathan
Nature genetics, 03/2004, Letnik: 36, Številka: 3Journal Article
With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the ∼13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5′ regulatory sequences.
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