Notch1胞内结构域慢病毒表达载体及干扰载体的构建

Objective To construct rat N1ICD lentiviral over-expression vector (LV-N1ICD) and N1ICD lentivirus interference vector (LV-N1ICD-shRNA. Methods With the rat cDNA as a template, the N1ICD fragment was amplified by PCR to construct pGC-FU-N1ICD-3Flag shuttle plasmid by directly clone. Four pairs of N1ICD-shRNA oligonucleotide sequences were synthesized to construct the GVC112-N1ICD-shRNA interference plasmid. pGC-FU-N1ICD-3Flag and GVC112 -N1ICD-shRNA plasmids were co-transfected into 293T cells to screen for the best interference plasmid in the 4 GVC112-N1ICD-shRNA plasmids by detecting Flag expression. pGC-FU-N1ICD-3Flag or GVC112-N1ICD-shRNA plasmid along with with pHelper 1.0 and pHelper 2.0 plasmids were co-transfect into 293T cells to package LV-N1ICD and LV-N1ICD-shRNA, and the virus titer was determined by real-time PCR and drug screening method, respectively. H9c2 cells infected with LV-N1ICD and LV-N1ICD-shRNA respectively were assessed for cell viability using CCK-8 assay. Results pGC-FU-N1ICD-3Flag and GVC112-N1ICD-shRNA plasmid were verified by PCR, gene sequencing and Western blotting. Co-transfection of the plasmids with pHelper 1.0, and pHelper 2.0 plasmids into 293T cells obtained high-titer LV-N1ICD and LV-N1ICD-shRNA. LV-N1ICD was capable of promoting the cell viability and LV-N1ICD-shRNA produced an opposite effect. Conclusion The vectors LV-N1ICD and LV-N1ICD-shRNA have been successfully constructed and packaged, which have the biological functions of Notch1 signaling.