Research On The Translation Control Of F9 And MEN1 Gene With Nonsense Mutation And Gene Readthrough

Premature termination codon (PTC) is a type of genetic or acquired defect in which an amino acid codon is substituted by a stop codon. One third of genetic inherited diseases involve a premature termination codon, and many cancers are also linked to a PTC:Hemophilia B is an X-linked recessive genetic disease that impairs the body’s ability to control blood clotting or coagulation and is caused by the mutation of coagulation factor gene F9. Nonsense mutations are counted to 11.1% in F9 gene mutations according to the type of mutations registered in hemophilia database. Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of tumors. As a tumor suppressor gene, endocrine tumors are caused by MEN1 mutation, which (>80%) are inactivating nonsense and frame shift mutations that lead to the premature termination codon.Classical therapies, such as surgery, radiotherapy and chemotherapy treatments advance constantly. Considerable interest has focused on PTC as potential targets for treatment with translational bypass therapy (TBT). Indeed, drugs can promote PTC readthrough, partially restoring the synthesis of full-length proteins.Aminoglycoside antibiotics such as G418 and non-aminoglycoside such as PTC 124 can promote PTC readthrough exhibit a wide range of efficiency. Encourage results have been obtained only in some cases, particularly for’good responder’mutations. PTCs are subject to levels of readthrough, depending on several factors such as drugs, context of PTC and nonsense mediate mRNA decay (NMD). In addition, a large number of molecules have been waited for investigation for their ability to induce PTC readthrough.Firstly, a dual fluorescent reporter system for screens of premature termination codon readthrough was constructed. It rely the feature of fluorescent protein coding region to express a fusion protein from pDsRed-EGFPmtag-. The system consists of the vector and its PTC mutant pDsRed-EGFPmtag-Y445X. The reporter described here contains DsRed upstream of EGFP such that DsRed and EGFP were expressed from one reading frames. The resulting expression of DsRed and EGFP proteins provided high sensitivity and a quantitative measure of the ratio of readthrough patterns. To quantify the ratio of readthrough events within individual cells in the flow cytometry analysis, and identify cells expressing different readthrough patterns within a mixed cell culture. After transfected COS7 cell with wild type vector and its mutant, the readthrough levels of mutants with nonsense mutations were increase significantly by treatment of PTC 124, and this effect was dose-dependent. The same results were confirmed by qRT-PCR, which EGFP target gene was amplified. In universal methods, such as a reporter of cell-based firefly luciferase (FLuc), the last step of the experiment is to dilute the medium with lysis buffer and add a very high concentration of the FLuc substrate luciferin. The luciferin essentially out competes the reversible inhibitor and produces a luminescent signal which is mistakenly attributed to facilitated readthrough of the PTC mutation. This dual fluorescent reporter assay, and the positive, readthrough data generated directly with incubated cells could reveal that PTC suppressor may exhibit genuine readthrough activity in spite of its interference in the FLuc assay system.Mono-chromatic reporters require all-or-none readthrough decisions for on or off expression of GFP because it is difficult to compare fluorescence intensity of different cells without an internal control. The single dichromatic reporter removes the variability associated with co-expression of two separate reporters that express different fluorescent proteins. Inclusion of a multiple cloning site (MCS) region of the appropriate size located between the DsRed and EGFP reading frames. A fragment could be embedded here and expressed with the fusion. So, different PTC patterns within the MCS region between the DsRed and EGFP reading frames could be measured sensitively.Secondly, the coding sequence of F9 gene was embedded in the multiple cloning site of the vector pDsRed-EGFPmtag-. According to the mutations registered in hemophilia database, Y22X^ E258X and E296X were chose as our nonsense mutation target to produce mutant vectors. COS7 cells were transfected with mutants. Real-time PCR and flow cytometry analysis showed that mRNA and protein levels of mutants with PTC 124 treatment were increased significantly. The readthrough levels of PTC 124 were higher than G418. The results were confirmed partially in function level by procoagulant activity kit for coagulation factor 9.Thirdly, focus on human multiple endocrine neoplasia type 1 (MEN1), our group reported that MEN1 gene with nonsense mutation was subjected to NMD by the strategy of minigene, and full length protein menin was restored by PTC 124. There is no directed evidence for the function restoring of menin. HeLa cells were transfected with siRNA plasmid pSIR-MEN1-10. Real-time PCR analysis showed that mRNA levels of Caspase 8, one of the target gene of MEN1, were down regulated with the knock down of MEN1. Flow cytometry analysis showed that apoptosis of cells with siRNA treatment were slowed down.Finally, we expanded the applications of dual fluorescent reporter system for screens of premature termination codon readthrough. We constructed a mini-gene of Factor IX (Mini-hF9-II), which contains three parts:exon 1-7+intron 7+exon 8. The mini-gene was cloned into the mammalian expression vector pCMV-3B. According to RT-PCR and Western Blot analysis, the mini-gene could work in cells, product normal transcripts and express full-length proteins. We’ll investigate the efficiency of readthrough by the EJC model of NMD when the mini-gene is embedded within the dual fluorescent reporter system.We isolated and characterised mesenchymal stem cells (MSCs) from human nucleus pulposus tissue. The MSC can be obtained and expanded from various tissues, and it has multi direction differentiation abilities. Transgenes can transfected MSC easily and expressed steadily, and the MSC also have little damage from transgenes. The mesenchymal stem cells were transfected preliminarily with the vector pDsRed-EGFPmtag-should pave the way to the rational design, construction of dual fluorescent reporter system MSC model and to the animal model for tracing and drug screening in vivo.With the continuing accumulation of insights into the dual fluorescent reporter system involved in PTC suppression, the field is poised to develop the mechanism of translation control, powerful new therapies and expand the usefulness of new compounds beyond treatments for rare genetic diseases to common afflictions such as cancer.