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    • 4. 发明申请
    • DISRUPTION OF EXPRESSION OF MULTIPLE ALLELES OF MAMMALIAN GENES
    • 破坏MAMMALIAN基因多态性的表达
    • WO1997018333A1
    • 1997-05-22
    • PCT/US1996018828
    • 1996-11-15
    • THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
    • THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITYLI, LiminCOHEN, Stanley, N.
    • C12Q01/68
    • G01N33/57415C07K14/4702C12N15/00C12Q1/68C12Q1/6886C12Q2600/118C12Q2600/136
    • Methods are provided for identifying a gene at a random chromosomal locus in the genome of a mammalian cell. The method involves inactivating one copy of the gene by integrating one DNA construct (knockout construct) in that gene copy. The knockout construct includes a positive selection marker region sequence and, in a 5' direction from the selection marker region sequence, a transcription initiation region sequence responsive to a transactivation factor, said transcription initiation region oriented for antisense RNA transcription in the direction away from the selection marker region sequence. The second copy of the gene is inactivated by transforming the cells with a second DNA construct (transactivation construct) containing a gene sequence for the transactivation factor which initiates antisense RNA transcription extending from the knockout construct into the chromosomal locus flanking the knockout construct at its 5' end. Inactivation of both gene copies may result in a change in cell phenotype distinguishable from the wild-type phenotype. Optionally, the wild-type phenotype can be regained by introducing a third construct that can inhibit antisense RNA transcription. A gene is provided associated with tumor susceptibility of mammalian cells, tsg 101.
    • 提供了用于鉴定哺乳动物细胞基因组中随机染色体基因座上的基因的方法。 该方法包括通过在该基因拷贝中整合一个DNA构建体(敲除构建体)来使基因的一个拷贝失活。 敲除构建体包括阳性选择标记区序列,并且在从选择标记区序列的5'方向上,对反式激活因子有反应的转录起始区序列,所述转录起始区在远离 选择标记区序列。 通过用包含反式激活因子的基因序列的第二个DNA构建体(反式激活构建体)转化细胞来灭活该基因的第二个拷贝,其启动从敲除构建体延伸到其敲除构建体侧翼的染色体基因座的5' ' 结束。 两种基因拷贝的失活可导致与野生型表型区分开的细胞表型的变化。 任选地,可以通过引入可抑制反义RNA转录的第三构建体来恢复野生型表型。 提供与哺乳动物细胞tsg 101的肿瘤易感性相关的基因。