Patent ID: 7692059

Claim:
A method for generating dominant-negative forms of bacterial toxins, comprising the steps of: a. misexpressing a wild-type bacterial toxin gene of interest in Drosophila flies, using a GAL4/UAS expression system; b. determining the phenotypic consequence of such misexpression, and selecting flies in which the bacterial toxin gene of interest is expressed; c. mutagenizing the wild-type bacterial toxin gene in the selected flies to produce carriers of a mutant bacterial toxinDN gene that differs in structure from the wild-type toxin gene by i. preparing DNA encoding full-length bacterial toxin, ii. inserting the toxin-encoding gene into the pUAS-vector iii. transforming into E. coli DH5ÿ, iv. testing for successful recombinants, v. purifying the pUAS-toxin DNA, vi. injecting the pUAS-toxin construct comprising a marked transgene into fly embryos without the marked transgene, isolating transformants comprising the marked transgene in F1 generation carrying a mutant bacterial toxinDN, and vii. selecting and establishing balanced transformed pUAS-toxinDN fly lines; and d. causing the mutant bacterial toxin gene to be expressed in the selected flies by expressing UAS-toxin in specific tissues using a set of GAL lines.