title: [Construction of the N1ICD lentiviral over-expression and interference vectors].
abstract: 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 syn- thesized 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.
  
  
  CONCLUSIONS
  The vectors LV-N1ICD and LV-N1ICD-shRNA have been successfully constructed and packaged, which have the biological functions of Notch1 signaling.
authors.affiliation: Department of Cardiac Surgery, The First Affiliated Hospital, Nanchang University, Nanchang 330006, China.E-mail: Dr_zhouxliang@163.com.
authors.forename: Xueliang
authors.initials: X
authors.lastname: Zhou
authors.forename: Jichun
authors.initials: J
authors.lastname: Liu
mesh_terms: D000818:Animals; D005822:Genetic Vectors; D016086:Lentivirus; D010957:Plasmids; D034741:RNA, Small Interfering; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering