Construction Of Cell Strain Containing Inducible Interference Of NMD Factor UPF1and SMG1Mediated By Lentiviral System

The cascade of events during gene expression, from transcription of the DNA encoded genetic information to the eventual protein synthesis, arguably represents some of the most influential biochemical pathways for a living organism. Naturally, diverse regulation mechanisms have evolved to ensure the accuracy of gene expression at multiple levels, amongst them is a process commonly referred to as nonsense-mediated mRNA decay (NMD) or mRNA surveillance.Nonsense-mediated mRNA decay (NMD) is one of a number of RNA surveillance pathways that help to ensure the fidelity of gene expression by degrading mRNAs. As the name suggests, NMD is responsible for recognizing and degrading mRNAs that contain premature termination codons (PTCs). The names coined to describe this PTC-associated mRNA turnover pathway, NMD and mRNA surveillance, emphasise its quality control func-tion in preventing the production of potentially deleterious C-terminally truncated proteins translated from PTC-containing mRNAs. Thus, NMD plays a key protective role in a long list of human diseases that are due to frameshift or nonsense mutations and result in the premature termination of mRNA translation, including β~0-thalassemia, cystic fibrosis, Duchennemuscular dystrophy and a number of cancers.The definition of aberrant transcription and start of NMD are determined by the protein complex binding on EJC, where UPF1and SMG1play key role for NMD path. UPF1is a RNA helicase and a RNA-dependent ATPase. SMG1is responsible for UPF1phosphorylation as a phosphatidylinositol3-kinase. RNA interference (RNAi) is a powerful genetic tool for loss-of-function studies in mammalian cells and is also considered a potentially powerful therapeutic modality for the treatment of a variety of human diseases. This is due to the very specific and potent degradation of the target RNA. Stable expression systems can be efficiently delivered to mammalian cells by viral vectors, resulting in long-term silencing of the target gene. In this study, we construct the pHIV-7-derived lentiviral vector pTIG (pHIV7-TetR-IRES-GFP) encoding a U6tetO-promoted short hairpin RNA (shRNA) cassette containing UPF1and SMGl. Target sites for hairpins were selected on the basis of a published design algorithm for19-base pair (bp) duplex siRNAs and nonstringent BLAST (Basic Local Alignment Search Tool) searches for uniqueness of the target sites. Then we synthesize double-stranded oligonucleotides of Smgl, Upf2and control siRNA.Most shRNA expression units rely on RNA polymerase III (pol-III) promoters such as the H1or U6promoter, we generated a lentiviral vector that contains one of the U6tetO-shRNA units and a CMV-driven TetR-IRES-GFP cassette.The shRNA constructs were cloned directionally into restriction sites of pFRT-U6tetO as double-stranded oligo cassettes. Finally we inserted U6tetO-shC, U6tetO-shS and U6tetO-shU into pTIG to create the all-in-one lentiviral vectors for inducible RNAi, pTIG-U6tetO-shC、pTIG-U6tetO-shS and pTIG-U6tetO-shU. Regulated RNAi systems include inducible pol-III promoters with tethered tetracycline operators (TetO) and co-expression of the tetracycline regulatory protein, TetR.In the absence of tetracycline or its derivative doxycycline (dox), TetR homodimer binds tightly to the Tet operon(s) inserted within the promoter and acts to suppress transcription. Upon its addition, tetracycline binds with high affinity to the Tet repressor homodimer in a1:1stoichiometry and causes a conformational change in the repressor that renders it unable to bind to TetO, allowing expression of the shRNA transcript.The resultant plasmids were transfected into HEK293T cell with the help of packaging plasmids to result in recombinant virus harboring hairpin RNA cassette. The resultant virus were then used to infect AD293cell to observe the expression of hairpin RNA gene. We have obtained the cell strains expressing hairpin RNA of UPF1and SMG1. These cell strains could be used for next research on the mechanism of NMD and gene screen.