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Methods of genetically encoding unnatural amino acids in eukaryotic cells using orthogonal tRNA/synthetase pairs
Methods of genetically encoding unnatural amino acids in eukaryotic cells using orthogonal tRNA/synthetase pairs
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机译:使用正交tRNA /合成酶对在真核细胞中遗传编码非天然氨基酸的方法
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摘要
This disclosure concerns compositions and methods for genetically encoding and expressing prokaryotic tRNAs in eukaryotic cells. In some embodiments, the disclosure concerns methods and compositions for expressing unnatural amino acids in eukaryotic cells using orthogonal tRNA/synthetase pairs. In certain embodiments, the methods involve expressing prokaryotic tRNA/synthetase pairs in eukaryotic cells, for instance mammalian cells or yeast cells (such as those that are NMD-deficient), under the control of a pol III promoter, for instance a type-3 pol III promoter or an internal leader promoter. Also provided are cell lines that are NMD-deficient and methods of increasing the efficiency of UAA incorporation in a cell that include de-activating the NMD pathway in the cell. Also provided are methods increasing the efficiency of incorporation of an unnatural amino acid in a cell by disrupting a Nonsense-Mediated mRNA Decay—(NMD) pathway in the cell.
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机译:本公开涉及用于在真核细胞中遗传编码和表达原核tRNA的组合物和方法。在一些实施方案中,本公开内容涉及使用正交tRNA /合成酶对在真核细胞中表达非天然氨基酸的方法和组合物。在某些实施方案中,所述方法包括在pol III启动子例如3型的控制下,在真核细胞,例如哺乳动物细胞或酵母细胞(例如NMD缺陷的那些)的真核细胞中表达原核tRNA /合成酶对。 pol III启动子或内部前导启动子。还提供了NMD缺陷的细胞系和增加UAA掺入细胞效率的方法,包括使细胞中的NMD途径失活。还提供了通过破坏细胞中的无义介导的mRNA衰变-(NMD)途径来增加细胞中非天然氨基酸掺入效率的方法。
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