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Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

ABSTRACT: As an important post-transcriptional regulatory machinery mediated by ?21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing ?-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced ?-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of ?-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.

SUBMITTER: Wang X

PROVIDER: S-EPMC6760290 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

Wang Xi X Yuan Chengfu C Huang Bo B Fan Jiaming J Feng Yixiao Y Li Alexander J AJ Zhang Bo B Lei Yan Y Ye Zhenyu Z Zhao Ling L Cao Daigui D Yang Lijuan L Wu Di D Chen Xian X Liu Bin B Wagstaff William W He Fang F Wu Xiaoxing X Luo Huaxiu H Zhang Jing J Zhang Meng M Haydon Rex C RC Luu Hue H HH Lee Michael J MJ Moriatis Wolf Jennifer J Huang Ailong A He Tong-Chuan TC Zeng Zongyue Z

ACS synthetic biology 20190910 9

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease B ...[more]

PMID: 31465214

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Project description:BackgroundThe discovery of RNA interference phenomenon (RNAi) and understanding of its mechanisms has revolutionized our views on many molecular processes in the living cell. Among the other, RNAi is involved in silencing of transposable elements and in inhibition of virus infection in various eukaryotic organisms. Recent experimental studies demonstrate few cases of viral replication suppression via complementary interactions between the mammalian small RNAs and viral transcripts.Presentation of the hypothesisIt was found that >50% of the human genome is transcribed in different cell types and that these transcripts are mainly not associated with known protein coding genes, but represent non-coding RNAs of unknown functions. We propose a hypothesis that mammalian DNAs encode thousands RNA motifs that may serve for antiviral protection. We also presume that the evolutional success of some groups of genomic repeats and, in particular, of transposable elements (TEs) may be due to their ability to provide antiviral RNA motifs to the host organism. Intense genomic repeat propagation into the genome would inevitably cause bidirectional transcription of these sequences, and the resulting double-stranded RNAs may be recognized and processed by the RNA interference enzymatic machinery. Provided that these processed target motifs may be complementary to viral transcripts, fixation of the repeats into the host genome may be of a considerable benefit to the host. It fits with our bioinformatical data revealing thousands of 21-28 bp long motifs identical between human DNA and human-pathogenic adenoviral and herpesviral genomes. Many of these motifs are transcribed in human cells, and the transcribed part grows proportionally to their lengths. Many such motifs are included in human TEs. For example, one 23 nt-long motif that is a part of human abundant Alu retrotransposon, shares sequence identity with eight human adenoviral genomes.Testing the hypothesisThis hypothesis could be tested on various mammalian species and viruses infecting mammalian cells.Implications of the hypothesisThis hypothesis proposes that mammalian organisms may use their own genomes as sources of thousands of putative interfering RNA motifs that can be recruited to repress intracellular pathogens like proliferating viruses.ReviewersThis article was reviewed by Eugene V. Koonin, Valerian V. Dolja and Yuri V. Shpakovski.

Dataset Information

Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

Publications

Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

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