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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Efficient TalEN-mediated gene knockout in livestock
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Efficient TalEN-mediated gene knockout in livestock

机译：TalEN介导的家畜基因敲除

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摘要

Transcription activator-like effector nucleases (TalENs) are programmable ? nucleases that join Fokl endonuclease with the modular DNA-binding domain of TalEs. although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TalENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TalEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored bialleiic modification. We also developed a simple transposon coselection strategy for TalEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TalEN-pair enabled isolation of colonies with mono- and bialleiic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TalEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TalEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and bialleiic mutations of the LDL receptor gene as models of familial hypercholes-terolemia. TalENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.
机译：转录激活因子样效应核酸酶（TalENs）是可编程的吗？将Fok1核酸内切酶与TalEs的模块DNA结合结构域结合的核酸酶。尽管锌指核酸酶能够进行多种基因组修饰，但由于胚胎注射，原代细胞培养的技术限制以及难以在有限的预算范围内生产可靠的试剂，它们在牲畜基因工程中的应用受到了限制。相反，我们发现TalENs可以轻松制造，超过一半（23/36，64％）的人在原代细胞中显示出高活性。 TalEN mRNA的细胞质注射到牲畜受精卵中能够诱导高达75％的受分析胚胎中的基因KO，其中一部分具有双烯丙基修饰。我们还开发了一种简单的转座子共选策略，用于TalEN介导的原代成纤维细胞中的基因修饰，既可以富集修饰细胞，又可以有效分离修饰菌落。用单个TalEN对处理后的共选能够分离出分别具有多达54％和17％菌落的具有单-和双烯丙基修饰的菌落。在用两个针对同一染色体的TalEN对进行共选择后，能够分离出具有较大染色体缺失和倒置的菌落（分别占菌落的10％和4％）。 TalEN修饰的Ossabaw猪胎儿成纤维细胞是克隆的有效核供体，从而产生了包含LDL受体基因的单和双烯丙基突变的微型猪，作为家族性高胆固醇血症的模型。因此，TalENs似乎代表了用于生物医学和农业应用的牲畜基因组修饰的高度便捷的平台。

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    Daniel F. Carlson; Wenfang Tan; Simon G. Lillico; Dana Stverakova; Chris Proudfoot; Michelle Christian; Daniel F. Voytas; Charles R. Long; C. Bruce A. Whitelaw; Scott C. Fahrenkrug;

  • 作者单位

    Center for Genome Engineering and University of Minnesota, St. Paul, MN 55108,Department of Animal Science, University of Minnesota, St. Paul, MN 55108,Recombinetics, Inc., St. Paul, MN 55114;

    Center for Genome Engineering and University of Minnesota, St. Paul, MN 55108,Department of Animal Science, University of Minnesota, St. Paul, MN 55108;

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, United Kingdom;

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, United Kingdom;

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, United Kingdom;

    Center for Genome Engineering and University of Minnesota, St. Paul, MN 55108,Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455;

    Center for Genome Engineering and University of Minnesota, St. Paul, MN 55108,Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455;

    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843;

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, United Kingdom;

    Center for Genome Engineering and University of Minnesota, St. Paul, MN 55108,Department of Animal Science, University of Minnesota, St. Paul, MN 55108,Recombinetics, Inc., St. Paul, MN 55114;

  • 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

    tal-effector nuclease; biotechnology; gene-editing;

    机译：tal效应核酸酶;生物技术基因编辑;

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