title: [Construction of recombinant adenovirus expressing shRNA targeting human  leukocyte-derived arginine aminopeptidase gene].
abstract: OBJECTIVE
  To construct the recombinant adenovirus expressing small hairpin RNA (shRNA) targeting human leukocyte-derived arginine aminopeptidase (LRAP) gene and silence the expression of LRAP in human retinal microvascular endothelial cells (HRMECs).
  
  
  METHODS
  Three pairs of oligonucleotides coding for shRNAs targeting human LRAP gene were designed and synthesized,  and were cloned into the shuttle vector pRNAT-H1.1/Adeno after  annealing. The three constructed shuttle plasmids,  through homologous recombination with adenoviral backbone vector pAdEasy-1,  were transformed into E.coli BJ5183-AD-  to produce recombinant adenoviral plasmids. And then the recombinants linearized with Pac I were transfected into 293a cells to package adenoviruses. After amplification and titter determination,  the recombinant adenoviruses were used to infect the primary HRMECs. Quantitative real-time PCR was taken to determine the relative amount of LRAP mRNA to screen the adenovirus with the highest silencing efficiency. The level of LRAP protein after RNA interference in HRMECs was further determined by Western blotting.
  
  
  RESULTS
  PCR,  restriction digestion and DNA sequencing confirmed that the purpose shRNA-coding sequences were correctly inserted into the shuttle vectors and adenoviral plasmids,  and the recombinant adenoviruses were packaged successfully in 293a cells. The most effective adenovirus   with the silencing efficiency up to 79% was selected by quantitative real-time PCR,  which significantly lowered the expression of LRAP in HRMECs.
  
  
  CONCLUSIONS
  The recombinant adenovirus expressing shRNA effectively silencing LRAP gene was constructed successfully,  which would facilitate further study of the role that LRAP plays in the development of diabetic retinopathy.
authors.affiliation: Sun Yat-sen University, Guangzhou, China. zengmzh@mail.sysu.edu.cn
authors.forename: Mei-zhen
authors.initials: MZ
authors.lastname: Zeng
authors.forename: Yan
authors.initials: Y
authors.lastname: Luo
authors.forename: Shao-fen
authors.initials: SF
authors.lastname: Lin
authors.forename: Xiao-yun
authors.initials: XY
authors.lastname: Chen
authors.forename: Jing-yi
authors.initials: JY
authors.lastname: Tian
authors.forename: Shi-bo
authors.initials: SB
authors.lastname: Tang
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000626:Aminopeptidases / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003930:Diabetic Retinopathy / Q000209:etiology; D042783:Endothelial Cells / Q000378:metabolism; D020868:Gene Silencing; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; D000626:Aminopeptidases; C503944:ERAP2 protein, human