title: Validating the use of short interfering RNA as a novel technique for cell-specific target gene knockdown in lung ischemia-reperfusion injury.
abstract: OBJECTIVE
  Short interfering RNA is an effective method for target gene knockdown. However, concerns surround the design, administration, efficacy, specificity, and immunostimulatory potential. Although uptake by alveolar macrophages has been demonstrated, studies have not examined its use in lung ischemia-reperfusion injury. We describe the validation of short interference RNA as a novel technique for cell-specific target gene knockdown in our model of lung ischemia-reperfusion injury.
  
  
  METHODS
  Dose-response experiments were performed, and 3 distinct sequences of toll-like receptor-4, toll-like receptor-2, and myeloid differentiation factor-88 short interference RNA were tested for efficacy of knockdown. Saline, lipid vector, and noncoding short interference RNA controls were used. Similar experiments were performed in primary cultures of resident pulmonary cells. Target protein knockdown was assessed by Western blot. Rat serum and cell culture media were assessed for interferon and cytokine production. Biotin labeling was used to assess short interference RNA uptake.
  
  
  RESULTS
  Target protein expression was significantly reduced using short interference RNA. However, toll-like receptor-4 knockdown was isolated to alveolar macrophages, and biotin labeling confirmed toll-like receptor-4 short interference RNA localization to alveolar macrophages. There was significant knockdown of toll-like receptor-4 expression in cultured cells treated with toll-like receptor-4 short interference RNA. There was no significant change in interferon production after short interference RNA treatment. There was effective target protein knockdown with each sequence used.
  
  
  CONCLUSIONS
  Short interference RNA is a valid method for achieving target protein knockdown in alveolar macrophages and is an important tool in the evaluation of its role in the development of lung ischemia-reperfusion injury.
authors.affiliation: Division of Thoracic Surgery, University of Washington, Seattle, Wash.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Merry
authors.affiliation: Division of Thoracic Surgery, University of Washington, Seattle, Wash.
authors.forename: Patrick
authors.initials: P
authors.lastname: Phelan
authors.affiliation: Division of Thoracic Surgery, University of Washington, Seattle, Wash; University of Washington, Center for Lung Biology, Seattle, Wash. Electronic address: bhwang@uw.edu.
authors.forename: Billanna
authors.initials: B
authors.lastname: Hwang
authors.affiliation: Division of Thoracic Surgery, University of Washington, Seattle, Wash; University of Washington, Center for Lung Biology, Seattle, Wash.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Mulligan
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D008168:Lung / Q000378:metabolism / Q000473:pathology; D055370:Lung Injury / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D020318:Rats, Long-Evans; D015427:Reperfusion Injury / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D052096:Warm Ischemia
chemical_list: C507781:Myd88 protein, rat; D053594:Myeloid Differentiation Factor 88; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495343:Tlr2 protein, rat; C495347:Tlr4 protein, rat; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4
keywords: TLR2; TLR4; lung reperfusion injury; siRNA