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    • 4. 发明申请
    • TETRACYCLINE-CONTROLLED INSERTION ELEMENT FOR SYSTEMATIC MUTAGENESIS
    • 用于系统MUTAGENESIS的TETRACYCLINE控制插入元件
    • WO2006084695A3
    • 2006-11-02
    • PCT/EP2006001163
    • 2006-02-09
    • UNIV FRIEDRICH ALEXANDER ERHILLEN WOLFGANGBERTRAM RALPHKOESTNER MARTIN
    • HILLEN WOLFGANGBERTRAM RALPHKOESTNER MARTIN
    • C12N15/82C12N15/71C12N15/74C12N15/85
    • C12N15/8216C12N15/635C12N2800/30C12N2800/90C12N2820/55C12N2830/003
    • 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.
    • 本发明涉及插入元件,其中所述插入元件是重组核酸分子,其包含在5' 3'排列:(i)首先重组结合位点; (ii)可操作地连接到启动子和任选地转录控制序列的抗性基因; (iii)四环素控制的启动子; 和(iv)第二重组结合位点,其中所述四环素受控启动子被取向以允许在第二重组结合位点的方向上转录。 本发明还涉及一种用于产生细胞的条件敲除突变体的方法,包括以下步骤:(a)将包含本发明的插入元件的核酸分子导入细胞中,其中插入 元素被转染到细胞中,伴随着纯化的重组; (b)将插入元件整合到细胞基因组中; 和(c)选择针对插入元件的抗性基因赋予抗性的底物的抗性的细胞; 其中在步骤(a)之前或伴随着步骤(a),将四环素敏感(多)肽的表达构建体引入细胞中,所述(多)肽能够结合插入元件的四环素受控启动子并调节 其表达。 最后,本发明涉及含有本发明的插入元件的细胞,鉴定鉴定病原体存活的遗传决定因素的方法以及产生转基因动物和植物的方法。