| The brain edema is one of the basic pathological changes of ischemic cerebrovascular disease(ICD), which is highly associated with the prognosis of ICD. However, the therapy for the brain edema is not very satisfying. Recent studies showed that AQP4 (aquaporin-4) played a key role in brain water transport, and the over-expression of AQP4 after the cerebral infarction is an inducing factor of the brain edema followed by cerebral infarction. Therefore, above facts suggests the inhibition of AQP4 is a new approach for treating the brain edema after the cerebral infarction.RNAi (RNA interference) technology, a new efficient and specific tool for gene knockdown, has developed rapidly in recent years and has a wide application prospect. Due to the structural particularity of central nervous system(CNS), many RNAi vectors can not get into CNS, which inhibits the function of RNAi to a large extent. Lentiviral vector (LV) derived from HIV-1 can infect the nondividing cells including neurons through integrating into the host, so it has a stable RNAi effect. In addition, LV has a high infective effect and low immunogenicity. All above characteristics make LV widely used in gene therapy and gene function study in nervous system.A recombinant lentiviral vector plasmid expressing rat AQP4-shRNA was constructed, and viral particle was packaged and concentrated in this experiment. We also titrated the viral particle through the puromycin screening. Our study provides a theoretical and experimental basis for the further studies about the role of AQP4 on the brain edema and the exploration of a more effective therapeutic approach for the brain edema, and establishes a new method of lentiviral titration. Object:1 .To construct a recombinant lentiviral vector plasmid PLKO. 1-puro-shAQP4, and tomake concentrated viral particle stably expressing rat AQP4-shRNA;2.To establish a direct and convenient method of puromycin screening for titratinglentivirus.Methods:1. The target siRNA specific to rat AQP4 gene was designed and converted into cDNA of shRNA (small hairpin RNA). The cDNA was synthesized and inserted into vector plasmid PLKO.1-puro which was linearized by restriction endonucleases Age I and EcoR I. The recombinant plasmid was transformed into competent E. coli. DH5αcells. The positive recombinant clones were selected by ampicillin medium agar and identified by MluI and BamHI and DNA sequencing.2. 293T cells were cotransfected by recombinant vector plasmid of PLKO.1-puro-shAQP4, packaging plasmid ofΔ8.2 and envelop plasmid of VSV-G with FUGENE6 transfection reagent to produce lentivirus particles, and the particles were concentrated by ultracentrifuge.3. Recombinant lentivirus were diluted by serial dilution, and infected Eca9706 cells, then titrated the virus with the method of the puromycin screening at the least fatal dose to Eca9706 cells.Results:1. Two target siRNAs selected from rat AQP4 gene mRNA were used to design 2 pairs of corresponding complementary oligonucleotide segments, and these complementary oligonucleotide segments were recombinated into lentivirus vector plasmid of PLKO.1-puro, then two positive recombinant colonies were selected.2. Restriction enzyme digestion and sequencing showed recombinant lentiviral vector plasmid PLKO.1-puro-shAQP4 was correct.3. Lentiviral particles were made through three plasmid cotransfection of 293T cells, and were concentrated by 100 folds.4. The least fatal dose of puromycin to Eca9706 was 2.5μg/ml, by which the concentrated virus was titrated 10~7TU/ml.Conclusion:1. Recombinant lentiviral vector plasmid with rat AQP4 shRNA were successfullyconstructed, and the packaging and titrating of lentiviral particle were completed, which provides a theoretical and experimental basis for the further studies on the roleof AQP4 in brain edema and exploration of a more effective therapeutic approach forbrain edema.2. For the first time, we established a direct and convenient method of titratinglentivirus with puromycin screening.
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