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    Efficient gene transfer into primary lymphocytes obviating the need for drug selection
    5.
    发明授权
    Efficient gene transfer into primary lymphocytes obviating the need for drug selection 失效
    有效的基因转移到原发性淋巴细胞中，避免了药物选择的需要

    公开(公告)号：US5686280A

    公开(公告)日：1997-11-11

    申请号：US302232

    申请日：1994-09-08

    申请人： Joseph Dougherty ,  Ming-ling Kuo ,  Natalie Sutkowski ,  Yacov Ron

    发明人： Joseph Dougherty ,  Ming-ling Kuo ,  Natalie Sutkowski ,  Yacov Ron

    IPC分类号： C12N15/09 ,  C12N5/10 ,  C12N15/85 ,  C12N15/867 ,  C12N15/87 ,  C12N15/64

    CPC分类号： C12N15/86 ,  C12N15/85 ,  C12N15/87 ,  C12N2740/13043

    摘要： The present invention pertains to a method for efficiently introducing exogenous genes into primary lymphoid cells without drug selection which comprises the steps (a) deriving a retroviral vector and a helper cell combination that will yield a level of virus production in the range from 5.times.10.sup.6 to 5.times.10.sup.7 units/ml by transfecting a vector into a helper cell followed by selection, isolation of cell clones, and determination of viral titers to identify which virus-producing cell lines produce a virus titer in the range from 5.times.10.sup.6 to 5.times.10.sup.7 units/ml; (b) isolating a lymphoid cell subpopulation which can repopulate a specific lymphoid lineage or is a long-lived population by treating a suspension of lymphoid cells with a monoclonal antibody which removes undesired lymphoid cells to obtain an enriched lymphoid subpopulation; (c) culturing the enriched lymphoid subpopulation from step (b) with growth factors specific to the lymphoid subpopulation; (d) co-cultivating the lymphoid subpopulation from step (c) with a lawn of irradiated virus-producing cell line from step (a) to produce an infected lymphoid subpopulation; and (e) harvesting the infected lymphoid subpopulation.

    摘要翻译： 本发明涉及一种有效地将外源基因引入到没有药物选择的原代淋巴细胞中的方法，其包括以下步骤：（a）导出逆转录病毒载体和辅助细胞组合，其将产生5×10 6至5×10 7 将载体转染到辅助细胞中，然后选择，分离细胞克隆，以及测定病毒滴度以鉴定哪些病毒产生细胞系产生5×10 6至5×10 7单位/ ml范围内的病毒效价; （b）通过用去除不需要的淋巴细胞以获得富集的淋巴亚群的单克隆抗体处理淋巴样细胞的悬浮液来分离能够重新形成特定淋巴系谱或长寿命群体的淋巴样细胞亚群; （c）用来自步骤（b）的富集淋巴样亚群培养具有淋巴亚群特异性的生长因子; （d）将来自步骤（c）的淋巴样亚群与来自步骤（a）的经辐照的产生病毒的细胞系的草坪共同培养以产生感染的淋巴样亚群; 和（e）收获感染的淋巴样亚群。

    Efficient gene transfer into primary lymphocytes obviating the need for drug selection
    6.
    发明授权
    Efficient gene transfer into primary lymphocytes obviating the need for drug selection 失效
    有效的基因转移到原发性淋巴细胞中，避免了药物选择的需要

    公开(公告)号：US5667998A

    公开(公告)日：1997-09-16

    申请号：US477363

    申请日：1995-06-07

    申请人： Joseph Dougherty ,  Ming-Ling Kuo ,  Natalie Sutkowski ,  Yacov Ron

    发明人： Joseph Dougherty ,  Ming-Ling Kuo ,  Natalie Sutkowski ,  Yacov Ron

    IPC分类号： C12N15/09 ,  C12N5/10 ,  C12N15/85 ,  C12N15/867 ,  C12N15/87 ,  C12N15/64

    CPC分类号： C12N15/86 ,  C12N15/85 ,  C12N15/87 ,  C12N2740/13043

    摘要： The present invention pertains to a method for efficiently introducing exogenous genes into primary lymphoid cells without drug selection which comprises the steps (a) deriving a retroviral vector and a helper cell combination that will yield a level of virus production in the range from 5.times.10.sup.6 to 5.times.10.sup.7 units/ml by transfecting a vector into a helper cell followed by selection, isolation of cell clones, and determination of viral titers to identify which virus-producing cell lines produce a virus titer in the range from 5.times.10.sup.6 to 5.times.10.sup.7 units/ml; (b) isolating a lymphoid cell subpopulation which can repopulate a specific lymphoid lineage or is a long-lived population by treating a suspension of lymphoid cells with a monoclonal antibody which removes undesired lymphoid cells to obtain an enriched lymphoid subpopulation; (c) culturing the enriched lymphoid subpopulation from step (b) with growth factors specific to the lymphoid subpopulation; (d) co-cultivating the lymphoid subpopulation from step (c) with a lawn of irradiated virus-producing cell line from step (a) to produce an infected lymphoid subpopulation; and (e) harvesting the infected lymphoid subpopulation. The invention further relates to a population of transfected lymphocytes, in which greater than about 90% of the lymphocytes contain a provirus.

    摘要翻译： 本发明涉及一种有效地将外源基因引入到没有药物选择的原代淋巴细胞中的方法，其包括以下步骤：（a）导出逆转录病毒载体和辅助细胞组合，其将产生5×10 6至5×10 7 将载体转染到辅助细胞中，然后选择，分离细胞克隆，以及测定病毒滴度以鉴定哪些病毒产生细胞系产生5×10 6至5×10 7单位/ ml范围内的病毒效价; （b）通过用去除不需要的淋巴细胞以获得富集的淋巴亚群的单克隆抗体处理淋巴样细胞的悬浮液来分离能够重新形成特定淋巴系谱或长寿命群体的淋巴样细胞亚群; （c）用来自步骤（b）的富集淋巴样亚群培养具有淋巴亚群特异性的生长因子; （d）将来自步骤（c）的淋巴样亚群与来自步骤（a）的经辐照的产生病毒的细胞系的草坪共同培养以产生感染的淋巴样亚群; 和（e）收获感染的淋巴样亚群。 本发明还涉及一组转染的淋巴细胞，其中大于约90％的淋巴细胞含有前病毒。