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In vitro or in vivo site-specific DNA recombination, useful e.g. for gene inactivation, using Cre recombinase that lacks heterologous protein-transduction domain

机译:体外或体内位点特异性DNA重组,例如使用缺少异源蛋白转导结构域的Cre重组酶进行基因灭活

摘要

Site-specific DNA recombination in eukaryotic cells in vitro comprising using Cre recombinase that lacks heterologous protein-transduction domains, is new. Site-specific DNA recombination in eukaryotic cells in vitro comprises: (a) introducing a polypeptide (I) with Cre recombinase activity but not containing any heterologous protein-transduction domains, into a eukaryotic cell culture where the cells contain DNA region(s) with recognition site(s) for (I); and (b) culturing the cells under suitable conditions for DNA recombination. Independent claims are also included for: (1) a polypeptide (Ia) with Cre recombinase activity comprising fully defined sequences of 343 amino acids (S2) or 371 amino acids (S4), as given in the specification, or their derivatives, homologues and active fragments, excluding the native enzyme from phage P1; (2) nucleic acid sequences (II) comprising fully defined sequences of 1032 base pairs (S1) or 1116 base pairs (S3), as given in the specification, encoding (I); (3) isolated cells that contain (Ia) or (II); (4) a reporter system for detecting site-specific DNA recombination in eukaryotic cells; (5) isolated cells containing the reporter system of (4); (6) preparing (Ia) by recombinant expression; (7) a kit for the new process or the method of (6); and (8) use of glycerol for performing or improving protein transduction.
机译:新的体外真核细胞中的位点特异性DNA重组是新的,包括使用缺少异源蛋白质转导结构域的Cre重组酶。体外真核细胞中的位点特异性DNA重组包括:(a)将具有Cre重组酶活性但不包含任何异源蛋白质转导域的多肽(I)引入真核细胞培养物中,其中细胞包含带有(I)的识别站点; (b)在合适的条件下培养细胞以进行DNA重组。还包括以下方面的独立权利要求:(1)具有Cre重组酶活性的多肽(Ia),包括本说明书中给出的343个氨基酸(S2)或371个氨基酸(S4)的完整定义的序列,或其衍生物,同源物和活性片段,不包括噬菌体P1的天然酶; (2)核酸序列(II),其包含说明书中给出的编码(I)的1032个碱基对(S1)或1116个碱基对(S3)的完全限定的序列; (3)分离的含有(Ia)或(II)的细胞; (4)用于检测真核细胞中位点特异性DNA重组的报告系统。 (5)分离的细胞,其包含(4)的报道分子系统; (6)通过重组表达制备(Ia); (7)用于(6)所述的新方法或方法的试剂盒; (8)甘油用于进行或改善蛋白质转导的用途。

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