Method for introducing non-natural amino acid coding system into cell line through Rosa26 site and cell line thereof

An unnatural amino acid, prosa26-ef1 technology, applied in the field of mouse embryonic stem cell lines, can solve the problems of easy silencing of repetitive sequences, uncertain targeting sites of transposons, non-uniformity, etc., to avoid repetitive sequences easily. The effect of being silenced and improving the introduction efficiency

CN113481239APending Publication Date: 2021-10-08WEST CHINA HOSPITAL SICHUAN UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2021-10-08

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Abstract

The invention discloses a method for introducing an unnatural amino acid coding system into a cell line through a Rosa26 site and the cell line thereof. The method comprises the following steps: (1) constructing plasmids containing multi-copy tRNA by utilizing tRNA promoters from different species; (2) constructing a plasmid which targets the Rose26 site and contains a modified aminoacyl tRNA synthetase system capable of recognizing different non-natural amino acids; and (3) constructing a mouse embryonic stem cell line in which a non-natural amino acid (Uaa) coding system is introduced at the Rosa26 site, and introducing Uaa into stem cells. According to the invention, tRNA promoters of different species are adopted, so that the problem that a repetitive sequence is easy to silence when the same promoter is used can be effectively avoided. Meanwhile, an aminoacyl tRNA synthetase system is introduced through a targeting Rose26 site, so that the defect of transposon random insertion is overcome, the cytotoxicity is reduced, and the introduction efficiency of non-natural amino acid is improved.
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