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首页> 外文学位 >Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells.
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Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells.

机译：增强哺乳动物细胞中质粒DNA稳定性的基因组DNA序列的分离和表征。

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摘要

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.; Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.; A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of ~0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.
机译：易于生产和操作使质粒DNA成为其在基因转移技术（例如基因治疗和DNA疫苗）中使用的主要靶标。然而，质粒的主要缺点是其在哺乳动物细胞中的稳定性。通常通过细胞机制或简单稀释导致的有丝分裂而丢失质粒DNA。该研究着手寻找能增强质粒DNA在哺乳动物细胞中稳定性的哺乳动物基因组DNA序列。创建基于质粒的基因组DNA文库，我们能够通过将文库转染到人胚肾（HEK 293）细胞中来筛选人基因组。使用G418选择含有质粒DNA的细胞14天。然后使用转化过程作为检测器，筛选所得的群体中是否存在生物活性质粒DNA。修改了市售质粒DNA分离试剂盒，以从哺乳动物细胞中提取质粒DNA。标准化协议的检测极限为一百万个细胞中每个细胞约0.6个质粒。这允许检测在HEK 293细胞中维持32天的45个质粒。与人类基因组相比，选定插入片段的测序显示胸腺嘧啶含量明显更高。具有高A / T含量的序列已与哺乳动物细胞中的支架/基质附着区（S / MAR）序列相关联。因此，哺乳动物细胞中质粒的稳定性可能需要与核基质缔合。

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  • 作者

    Manocha, Marcus M. S.;

  • 作者单位

    Brock University (Canada).;

  • 授予单位 Brock University (Canada).;
  • 学科 Biology Genetics.; Biology Cell.
  • 学位 M.Sc.
  • 年度 2006
  • 页码 144 p.
  • 总页数 144
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 遗传学;细胞生物学;
  • 关键词

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