title: Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models.
abstract: BACKGROUND
  Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process.
  
  
  RESULTS
  To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous beta-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.
  
  
  CONCLUSIONS
  We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.
authors.affiliation: Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London, SW7 2AZ, UK. ramalho@imagem.ibili.uc.pt
authors.forename: José S
authors.initials: JS
authors.lastname: Ramalho
authors.forename: Ross
authors.initials: R
authors.lastname: Anders
authors.forename: Gesine B
authors.initials: GB
authors.lastname: Jaissle
authors.forename: Mathias W
authors.initials: MW
authors.lastname: Seeliger
authors.forename: Clare
authors.initials: C
authors.lastname: Huxley
authors.forename: Miguel C
authors.initials: MC
authors.lastname: Seabra
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D015794:Choroideremia / Q000235:genetics* / Q000378:metabolism*; D003433:Crosses, Genetic; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D005799:Genes, Dominant / Q000235:genetics*; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D006200:Hair Color / Q000235:genetics / Q000502:physiology; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012160:Retina / Q000503:physiopathology; D014785:Vision, Ocular / Q000235:genetics / Q000502:physiology; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
chemical_list: D012333:RNA, Messenger; D000075745:rab27 GTP-Binding Proteins; C512958:Rab27b protein, mouse; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins