From clone to mutant gene

Abstract

This chapter reviews several methods for isolating mutations in cloned Drosophila genes and provide protocols for generating P-element-induced mutations at targeted loci. In these protocols, local transposition of P-elements is used to increase the probability of insertion into the site of interest. The chapter also describes flanking sequence rescue screens to identify lines bearing insertions into the gene of interest from collections of P-element transposants. Several properties of P-elements make them attractive as mutagenic agents for targeted genetic screens. Recombinant P-element derivatives whose mobility is controlled by mating to a source of transposase and whose transposition is selectable by phenotype are widely available, making the generation of single or multiple hit transposants straightforward. Because the P-element transpositions tag the inserted site with a known DNA sequence, insertions into the target region can be detected at the DNA level without a prediction of phenotype. P-element insertions isolated in or near a gene of interest can be used as substrates to generate new alleles at very high efficiencies.

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