TETRACYCLINE-CONTROLLED INSERTION ELEMENT FOR SYSTEMATIC MUTAGENESIS

摘要

The present invention relates to an insertion element, wherein said insertion element is a recombinant nucleic acid molecule which comprises, in 5' ? 3' arrangement: (i) a first recombines binding site; (ii) a resistance gene, operatively linked to a promoter and, optionally, to transcription control sequences; (iii) a tetracycline-controlled promoter; and (iv) a second recombines binding site, wherein said tetracycline-controlled promoter is oriented to allow transcription in direction of the second recombines binding site. The present invention also relates to a method for the generation of a conditional knock-out mutant of a cell, comprising the steps of: (a) introducing a nucleic acid molecule comprising the insertion element of the present invention into a cell, wherein the insertion element is transfected into the cell concomitant with purified recombines; (b) integrating the insertion element into the cellular genome; and (c) selecting cells for resistance against a substrate for which the resistance gene of the insertion element confers resistance; wherein prior to or concomitant with step (a) an expression construct for a tetracycline-sensitive (poly)peptide is introduced into the cell, said (poly)peptide being capable of binding to the tetracycline-controlled promoter of the insertion element and of modulating its expression. Finally, the present invention relates to a cell containing the insertion element of the present invention, to a method for identifying genetic determinants of pathogen survival and to a method for the generation of a transgenic animal and plant.