title: [Establishment of pancreatic ductal cell lines with stably silencing of MyD88 mediated by lentivirus].
abstract: OBJECTIVE
  To design and construct a lentiviral vector containing shRNA against rat myeloid differentiation protein 88 gene (MyD88), and to establish rat pancreatic ductal cell line with stable knockdown of MyD88 expression.
  
  
  METHODS
  Constructed two plasmid expression vectors coding shRNA against MyD88 and converted them into lentiviral particles using three-plamid package system, named as Lenti-sh MyD88-1 and LentishMyD88-2. Rat pancreatic ductal cell, ARIP were divided into untreated group, Lenti-Non Target group, LentishMyD88-1 and LentishMyD88-2 treated groups, and transduced with corresponding lentiviral particles. The GFP positive cells were selected by fluorescence-activated cell sorting and puromycin, the MyD88 gene silencing efficiency was detected by real-time RT-PCR.
  
  
  RESULTS
  After the transduction, we observed highly efficient transduction (reach to 100%) of lentivirus in ARIP cells by fluorescent microscopy and FACS. Quantitative RT-PCR showed that Lenti-shMyD88-1 reduced MyD88 mRNA expression by about 82.73% +/- 1.203% in ARIP cells, and LentishMyD88-2, reduced 75.56% +/- 1.176% as compared with that of the untreated cells (P < 0.05).
  
  
  CONCLUSIONS
  The results demonstrated that lentivector containing the short hairpin RNA expression cassette specifically targeting MyD88 (pLVshMyD88-1,-2) were successfully constructed, which could stably knock down the MyD88 expression in ARIP cells. This study finally provided a new and stable cell model for the study of MyD88's function in acute pancreatitis.
authors.affiliation: State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Ke-Ling
authors.initials: KL
authors.lastname: Chen
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Zhan
authors.initials: Z
authors.lastname: Lan
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.forename: Zong-Guang
authors.initials: ZG
authors.lastname: Zhou
mesh_terms: D000208:Acute Disease; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics*; D010183:Pancreatic Ducts / Q000166:cytology*; D010195:Pancreatitis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats
chemical_list: D053594:Myeloid Differentiation Factor 88; D012333:RNA, Messenger; D034741:RNA, Small Interfering