title: [Establishment of pancreatic acinar cell line AR42J with stable knockdown of GRP78].
abstract: OBJECTIVE
  To design and construct a lentiviral vector containing shRNA against rat glucose-regulated protein 78 gene (GRP78), and to establish rat pancreatic acinar cell line with stable knockdown of GRP78 expression.
  
  
  METHODS
  Constructed three plasmid expression vectors coding shRNA against GRP78, and converted them into lentiviral particles using three plasmid package systems. Then, AR42J cells were transduced with produced lentiviral particles. The green fluorescent protein (GFP) positive cells were selected by fluorescence-activated cell sorting (FACS), the GRP78 gene mRNA and protein expression were detected by real-time PCR and Western blot.
  
  
  RESULTS
  The DNA sequencing showed that the lentiviral vectors containing shRNA against GRP78 gene were constructed correctly. After the transduction, highly efficient transduction (> 85%) of lentivirus in AR42J cells was observed by fluorescent microscopy and FACS. Quantitative real-time PCR showed that GRP78 mRNA expression in AR42J cells was suppressed by LVshGRP78-1, LVshGRP78-2 and LVshGRP78-3 lentivirus about 69.4% +/- 1.42%, 74.7% +/- 1.69% and 86.6% +/- 1.73% as compared with that of the untreated cells (P < 0.05), while LV-Non Target had no significant effect on the GRP78 mRNA level (P > 0.05). Western blot showed that the suppressed effect of LVshGRP78 lentivirus on GRP78 protein was consistent with GRP78 mRNA.
  
  
  CONCLUSIONS
  The results demonstrated that lentiviral vectors containing the shRNA against GRP78 gene were successfully constructed, which could stably knock down the GRP78 expression in AR42J cells. This study will provide a new cell model for further study of the role of GRP78 in the pathogenesis of acute pancreatitis.
authors.affiliation: Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu 610041, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.forename: Ke-Ling
authors.initials: KL
authors.lastname: Chen
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Lie
authors.initials: L
authors.lastname: Yang
authors.forename: Hui
authors.initials: H
authors.lastname: Yan
authors.forename: Zong-Guang
authors.initials: ZG
authors.lastname: Zhou
mesh_terms: D061354:Acinar Cells / Q000166:cytology*; D000208:Acute Disease; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology*; D010195:Pancreatitis / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D014161:Transduction, Genetic; D000091342:Endoplasmic Reticulum Chaperone BiP
chemical_list: D006360:Heat-Shock Proteins; D034741:RNA, Small Interfering; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP