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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Protein transduction from retroviral Gag precursors
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Protein transduction from retroviral Gag precursors

机译：逆转录病毒Gag前体的蛋白转导

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摘要

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of non-retroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogram-ming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.
机译：逆转录病毒颗粒组装了数千个Gag多蛋白。由逆转录病毒蛋白酶介导的蛋白水解切割在细胞进入之前形成了生物活性的逆转录病毒蛋白亚基。我们假设该过程可用于非逆转录病毒蛋白向培养细胞的靶向，瞬时和剂量控制的转导。我们证明，γ-逆转录病毒颗粒可在其Gag或Gag-Pol前体的几个位置上耐受外源蛋白的掺入。细胞接触后数分钟内，受体介导的工程颗粒的吸收以及因此可能发生的细胞特异性摄取。非逆转录病毒蛋白递送的剂量和动力学取决于多蛋白前体中的位置。将含有核定位信号的蛋白质掺入逆转录病毒颗粒中，并通过逆转录病毒蛋白酶从前体中释放目标蛋白质，从而识别出工程化的靶位点。与整合缺陷型慢病毒载体相反，逆转录病毒多蛋白前体的蛋白转导是完全瞬时的，因为可以产生不将基因转导至靶细胞的蛋白转导逆转录病毒样颗粒。或者，产生了双功能蛋白递送颗粒制剂，其保持了其用作逆转录病毒转基因载体的能力。我们通过传递位点特异性DNA重组酶Flp显示了这种方法对诱导多能干细胞的靶向基因组工程的潜力。从Gag的基质位置蛋白水解释放Flp后的蛋白转导允许切除慢病毒转导的盒，该盒同时表达规范性重编程转录因子（Oct4，Klf4，Sox2，c-Myc）和荧光标记基因，从而产生诱导型多能干细胞，不含慢病毒转导的重编程基因。

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  • 作者

    Christine Voelkel; Melanie Galla; Tobias Maetzig; Eva Warlich; Johannes Kuehle; Daniela Zychlinski; Juergen Bode; Tobias Cantz; Axel Schambach; Christopher Baum;

  • 作者单位

    Department of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnResearch Group Stem Cell Biology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

    rnDepartment of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    flp recombinase; murine leukemia virus; pluripotent cells; protein transfer;

    机译：flp重组酶;鼠白血病病毒;多能细胞;蛋白质转移;

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