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    • 10. 发明申请
    • CONTROL OF ENDOGENOUS DNMT1 GENE EXPRESSION BY EXOGENOUS BINARY REGULATORY SYSTEMS
    • 异源二进制调节系统内源DNMT1基因表达的控制
    • US20110016548A1
    • 2011-01-20
    • US12838300
    • 2010-07-16
    • PETER W. LAIRDKWANGHO LEE
    • PETER W. LAIRDKWANGHO LEE
    • C12N15/87C12N15/63C12N5/10C07K2/00C07K14/00C07K14/035C07H21/04
    • A61K31/353Y02A50/401
    • Provided are methods for controlling endogenous gene expression comprising control of the DNA methyltransferase (Dnmt1) for modulation of DNA methylation and epigenetic mechanisms. Provided are transcriptional regulatory systems involving multiple (e.g., three) exogenous binary systems, lacI, tetR and Gal4, for reversible up/down regulation of endogenous target genes Provided are lac operator and repressor modifications for improved repression relative to wild type (WT) lac and tet systems. Provided are endogenous Dnmt1 promoter modifications, comprising targeted lac operator sequences that do not significantly alter promoter activity absent repressors, yet show substantially reduced expression of the targeted allele upon lac repressor introduction. The lacO targeted Dnmt1 allele is introducible into the mouse germline, to provide a respective upregulatable transcriptional control system in vivo (e.g., two binary systems, tet operator/tetVP16 and Gal4 binding sequence/Gal4VP16, and ES cell gene targeting experiments are conducted with a Dnmt1 promoter construct combining all three cis elements.
    • 提供了用于控制内源基因表达的方法,包括用于调节DNA甲基化和表观遗传机制的DNA甲基转移酶(Dnmt1)的控制。 提供了涉及多个(例如三个)外源二元体系(lacI,tetR和Gal4)用于内源性靶基因的可逆上/下调节的转录调控系统。提供了用于改善相对于野生型(WT)lac的抑制的lac操纵子和阻遏物修饰 和tet系统。 提供内源性Dnmt1启动子修饰,其包含不显着改变启动子活性的靶向lac操纵子序列,不存在阻遏物,并且在lac阻抑物引入时显示目标等位基因的表达显着降低。 将lacO靶向的Dnmt1等位基因导入小鼠种系,以在体内提供相应的可上调的转录控制系统(例如,两个二元系统,tet操纵子/ tetVP16和Gal4结合序列/ Gal4VP16,并且用a细胞基因靶向实验用 结合所有三个顺式元件的Dnmt1启动子构建体。