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    • 9. 发明申请
    • TETRACYCLINE-CONTROLLED INSERTION ELEMENT FOR SYSTEMATIC MUTAGENESIS
    • 用于系统性诱变的四环素控制插入元件
    • WO2006084695A2
    • 2006-08-17
    • PCT/EP2006/001163
    • 2006-02-09
    • FRIEDRICH-ALEXANDER UNIVERSITÄT ERLANGEN-NÜRNBERGHILLEN, WolfgangBERTRAM, RalphKÖSTNER, Martin
    • HILLEN, WolfgangBERTRAM, RalphKÖSTNER, 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.
    • 本发明涉及一种插入元件,其中所述插入元件是重组核酸分子,其以5'→3'排列包含:(i)第一重组结合位点; (ii)与启动子可操作地连接的抗性基因,并且任选地与转录控制序列连接; (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定向为允许在第二重组结合位点的方向转录。 本发明还涉及用于产生细胞的条件性敲除突变体的方法,其包括以下步骤:(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 元件与纯化的重组体一起转染到细胞中; (b)将插入元件整合到细胞基因组中; 和(c)选择细胞用于抗插入元件的抗性基因赋予其抗性的底物的抗性; 其中在步骤(a)之前或伴随步骤(a)将用于四环素敏感(多)肽的表达构建体引入细胞中,所述(多)肽能够与插入元件的四环素控制的启动子结合并且能够调节 它的表达。 最后,本发明涉及含有本发明的插入元件的细胞,鉴定病原体存活的遗传决定子的方法以及产生转基因动物和植物的方法。