title: [Effect of lentivirus-mediated APP695 RNAi on Aβ level in cortical neurons of APP695 transgenic mice].
abstract: OBJECTIVE
  To explore the effect of lentivirus-mediated APP695 RNAi on Aβ level in the cortical neurons of APP695 transgenic mice.
  
  
  METHODS
  The pFU-GW-iRNA plasmids expressing the RNAi sequences of human APP695 gene were confirmed by PCR (polymerase chain reaction) and sequencing. The recombined plasmids in combination with pHelper 1.0 and Helper 2.0 plasmids were transfected into 293T cells so as to construct the lentiviral vectors of RNA interference of APP695 gene. Virus in supernatant was collected and the viral titer measured. The cortical neurons of APP695 transgenic mice were infected with the packaged lentivirus. And it was designated as APP695-RNAi group. Cells infected with APP695-NC was designated as NC group and non-infected cells as CON group. The levels of APP695 mRNA were detected by real-time quantitative PCR. The APP695 protein was detected by Western blot and the levels of Aβ40 and Aβ402 were measured by ELlSA (enzyme-linked immunosorbent assay).
  
  
  RESULTS
  PCR and DNA sequencing demonstrated that the inserted sequences of APP695 shRNA were correct. The viral titer was 5 × 10(8) TU/ml. After the infection of APP695 shRNA recombinant lentivirus, the expression level of APP695 mRNA in the cortical neurons of APP695 transgenic mice decreased significantly (inhibition rate: 76.70%) versus NC and CON groups (P < 0.001). The lowered expression of APP695 proteins was consistent with APP695 mRNA. The levels of Aβ40 were (184 ± 15) ng/L, (647 ± 30) ng/L and (656 ± 40) ng/L in APP695-RNAi, NC and CON groups respectively. As compared with NC and CON groups, the levels of Aβ40 in APP695-RNAi group decreased significantly (P < 0.001). The levels of Aβ42 were (19.2 ± 1. 9) ng/L, (67.6 ± 6.0) ng/L and (68.6 ± 7.0) ng/L in APP695-RNAi, NC and CON groups respectively. As compared with NC and CON groups, the levels of Aβ42 decreased significantly in APP695-RNAi group(P < 0.001).
  
  
  CONCLUSIONS
  The inhibition of APP695 by lentivirus-mediated RNAi effectively decreases the levels of Aβ40 and Aβ42 in the cortical neurons of APP695 transgenic mice.
authors.affiliation: Department of Neurology, Zhengzhou University, Zhengzhou 450052, China.
authors.forename: Xin-yu
authors.initials: XY
authors.lastname: Zhao
authors.forename: Li-jun
authors.initials: LJ
authors.lastname: Wen
authors.forename: Guo-dong
authors.initials: GD
authors.lastname: Li
authors.forename: Zhi-qiang
authors.initials: ZQ
authors.lastname: Han
authors.forename: Bo
authors.initials: B
authors.lastname: Song
authors.forename: Yu-ming
authors.initials: YM
authors.lastname: Xu
mesh_terms: D016229:Amyloid beta-Peptides / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D016229:Amyloid beta-Peptides; D012333:RNA, Messenger