Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome

Abstract

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

Targeted gene alteration in Caenorhabditis elegans by gene conversion

“…Finally, the presence of a transposon at the target site may not be strictly required for gene conversion: an apparent instance of transposon-independent gene conversion has been obtained for the gene rol-6, albeit at an extremely low frequency 30 . An expansion of the available transposon insertion libraries 11-13 could conceivably tag every gene in C. elegans with a transposon and function as a basis for the detailed in vivo analysis of all genes by mutagenesis, as is currently underway in D. melanogaster 14,15 . In addition, intergenic regions that are not accessible by classical genetic analysis could be readily targeted.…”

“…An increase in these efforts could establish a C. elegans genome-wide transposon library. Such a project has been reported in Drosophila melanogaster, for which 450% of the genome has already been covered, and improved transposon-based techniques have been developed to address problems such as the nonrandom insertion bias of the transposons 14,15 .…”

Toward a complete Drosophiladeficiency kit

“…The principle behind the system is illustrated in Figure 1 and the FRT -bearing RS P elements developed for these elegant genome engineering approaches were used by the European DrosDel consortium to generate a collection of insertions for building a set of new deletions [6]. Using a conceptually similar approach, in this case with FRT sites carried on a set of piggyBac ( WH and RB ) and P ( XP ) transposon vectors, Exelixis Inc. also created a collection of new deletions [7]. In both cases, the deletions based on recombination between mapped FRT sites have their breakpoints located on the genome sequence with nucleotide resolution and thus the problem of precisely defining the extent of each deletion is solved.…”

Electrophysiological Recording From <em>Drosophila</em> Labellar Taste Sensilla

“…The relatively small number of neurons and the amenability to molecular and behavioral analysis make the Drosophila taste system an excellent model for the investigation of gustatory systems in general. The relative ease with which the system can be manipulated via genetic mutation or the GAL4-UAS expression system also serves as a valuable tool 14,15 .…”