| 曲妍佳,李晓宁,张萌萌,范雅婷,肖君华,李 凯,周宇荀.成簇pri-miRNAs表达载体的构建及表达验证[J].,2018,(20):3805-3811 |
| 成簇pri-miRNAs表达载体的构建及表达验证 |
| Construction and Expression Validation of Clustered pri-miRNAs Expression Vectors |
| 投稿时间：2018-02-18 修订日期：2018-04-12 |
| DOI：10.13241/j.cnki.pmb.2018.20.002 |
| 中文关键词: pri-miRNA miR29家族 多重表达载体 PTEN |
| 英文关键词: Pri-miRNA miR29 family Multiple expression vector PTEN |
| 基金项目:上海市科委实验动物专项基金项目(15140900500\16140901302)； 中央高校专项基金项目(CUSF-DH-D-2017054) |
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| 中文摘要: |
| 摘要 目的：旨在建立一个多重表达任意miRNA的方案，实现多个pri-miRNA表达阅读框的串联，用于miRNA簇或者miRNA家族的功能研究。方法：通过改造CRISPR/Cas9系统共表达载体42230，插入EGFP编码序列以及单个pri-miRNA序列，实现单个pri-miRNA表达载体可视化的构建。在单个pri-miRNA表达载体的基础上，通过同源重组的方式，插入下一个pri-miRNA序列，实现多个pri-miRNA表达阅读框的串联表达。结果：以miR29 家族的三个miRNA为例，验证了在pri-miRNA表达阅读框串联方面的可行性；在多重的pri-miRNA表达载体中，miRNA成熟体验证结果表明，pri-miR29a和pri-miR29b能够在哺乳动物细胞中被加工成熟，其成熟体明显地过表达。在转染的293A细胞中，miR29家族的靶基因PTEN的表达水平显著降低。结论：所构建的pri-miRNA多重表达方案，能够实现多个外源miRNA在同一细胞中的共表达，对于探索miRNA家族和miRNA簇的拮抗或协同调控功能起到重要的推进作用。 |
| 英文摘要: |
| ABSTRACT Objective: In order to create a protocol for multiple expression of arbitrary miRNAs in tandem with multiple pri-miRNA expression reading frames for the functional study of miRNA clusters or miRNA families. Methods: By modifying the CRISPR/Cas9 system co-expression vector 42230, the EGFP coding sequence and a single pri-miRNA sequence were inserted to enable visualization of a single pri-miRNA expression. Based on a single pri-miRNA expression vector, the next pri-miRNA sequence was inserted by homologous recombination to achieve tandem expression of multiple pri-miRNA expression reading frames. Results: Taking three miRNAs of the miR29 family as an example, the feasibility of pri-miRNA expression in reading frame tandem connection was verified. In multiple pri-miRNA expression vectors, miRNA maturation assay results showed that pri-miR29a and pri-miR29b were able to be processed to mature in mammalian cells and their mature bodies were significantly overexpressed. In transfected 293A cells, target gene PTEN of miR29 family expression level was significantly reduced. Conclusion: The pri-miRNA multiple expression protocol that we constructed enables the co-expression of multiple exogenous miRNAs in the same cell. It plays an important role in exploring the antagonistic or cooperative regulation of miRNA family and miRNA cluster. |
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