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    • 5. 发明授权
    • High efficiency retroviral vectors that contain none of viral coding sequences
    • 不含病毒编码序列的高效率逆转录病毒载体
    • US06451595B1
    • 2002-09-17
    • US09463067
    • 2000-01-14
    • Sunyoung KimSeung Shin YuJong-mook Kim
    • Sunyoung KimSeung Shin YuJong-mook Kim
    • C12N15867
    • C12N15/86C12N2740/13043C12N2830/42C12N2830/50C12N2840/203C12N2840/44
    • The present invention relates to improved retroviral vectors for gene therapy. In this invention, retroviral vectors with higher safety and efficiency are constructed from MLV-based starting vectors, MON and MIN. The improved vectors have the following features: 1) sequences corresponding to MLV-derived pol gene are completely deleted in the vectors, avoiding homologous recombination which has been a baffling problem in conventional retroviral vectors, 2) a heterologous intron, splicing acceptor and/or non-coding sequence are/is inserted into the upstream position of a cloning site, maximizing the expression of a foreign gene through efficient splicing, 3) the vectors contain either the full-length U3 sequence of 5′ LTR or a strong heterologous promoter instead, permitting the abundant production of RNA, 4) either IRES (Internal Ribosomal Entry Site) or internal SV40 minimal promoter is inserted into the downstream position of cloning site, enabling the simultaneous expression of two or more foreign genes. Since the improved retroviral vectors of this invention turn out to be safe and to express the foreign gene efficiently, they are useful for gene therapy and the like.
    • 本发明涉及用于基因治疗的改良逆转录病毒载体。 在本发明中,具有更高安全性和效率的逆转录病毒载体由基于MLV的起始载体MON和MIN构建。 改进的载体具有以下特征:1)对应于MLV衍生的pol基因的序列在载体中完全缺失,避免了在常规逆转录病毒载体中已经成为障碍问题的同源重组,2)异源内含子,剪接受体和/或 非编码序列被插入到克隆位点的上游位置,通过有效剪接使外源基因的表达最大化,3)载体含有5'LTR的全长U3序列或者强异源启动子 ,允许大量产生RNA,4)将IRES(内部核糖体进入位点)或内部SV40最小启动子插入到克隆位点的下游位置,使得能够同时表达两个或多个外源基因。 由于本发明改良的逆转录病毒载体是安全的并有效表达外源基因,所以它们可用于基因治疗等。
    • 8. 发明申请
    • METHOD FOR SELECTING A HIGH EXPRESSION RECOMBINANT CELL LINE
    • 选择高表达重组细胞系的方法
    • US20120009682A1
    • 2012-01-12
    • US13255004
    • 2010-03-30
    • MyungSam ChoMin Seok ChangJong-Mook KimHyunJoo LeeYoo Cheol SongManSu Kim
    • MyungSam ChoMin Seok ChangJong-Mook KimHyunJoo LeeYoo Cheol SongManSu Kim
    • C12N15/64C12N5/10C12N5/16C12N15/63
    • C12N15/65C12N15/1086C12N15/69C12N15/85C12N2800/24C12N2830/46C12N2830/50C12P21/02
    • The present invention relates to a method of selecting high producer clones by using an expression vector, the expression vector comprising: (i) a gene expression cassette comprising a selectable marker gene to which polyA has been inoperably linked; and (ii) a gene expression cassette which encodes a recombinant protein of interest and to which polyA has been operably linked. According to the invention, high producer clones can be selected from cell populations at least 10 times fewer than in the existing methods of selecting cell lines. Particularly, high producer clones can be selected using a low concentration of MTX compared to a conventional stepwise gene amplification strategy which comprises carrying out multiple amplification steps while increasing the concentration of MTX. Accordingly, the development period of cell lines can be shortened and the labor and cost required for selection of high-productivity cell clones can be reduced, whereby more efficient production of proteins is possible even when general selectable marker genes other than MTX are used.
    • 本发明涉及通过使用表达载体选择高生产者克隆的方法,所述表达载体包含:(i)包含聚A不可操作连接的选择标记基因的基因表达盒; 和(ii)编码目的重组蛋白质并且polyA已被可操作地连接的基因表达盒。 根据本发明,可以从细胞群体中选择高生产者克隆比选择细胞系的现有方法的至少10倍。 特别地,与传统的逐步基因扩增策略相比,可以使用低浓度的MTX来选择高生产者克隆,其包括在增加MTX浓度的同时进行多个扩增步骤。 因此,可以缩短细胞系的发育期,并且可以降低选择高生产率细胞克隆所需的劳动力和成本,从而即使使用MTX以外的一般可选择标记基因,蛋白质的更高效生产也是可能的。