title: U6 promoter-enhanced GlnUAG suppressor tRNA has higher suppression efficacy and can be stably expressed in 293 cells.
abstract: BACKGROUND
  Almost one-third of all human genetic diseases are the result of nonsense mutations that can result in truncated proteins. Nonsense suppressor tRNAs (NSTs) were proposed as valuable tools for gene therapy of genetic diseases caused by premature termination codons (PTCs). Although various strategies have been adapted aiming to increase NST expression and efficacy, low suppression efficacies of NSTs and toxicity associated with stable expression of suppressor tRNAs have hampered the development of NST-mediated gene therapy.
  
  
  METHODS
  We have employed the U6 promoter to enhance Gln-Amber suppressor tRNA (GlnUAG) expression and to increase PTC suppression in mammalian cells. In an attempt to study the toxic effects of NSTs, a stable 293 cell line constitutively expressing a U6 promoter-enhanced GlnUAG tRNA was established. To examine whether any proteomic changes occurred in cells that constitutively express suppressor tRNA, whole cell proteins from cells with and without any suppressor tRNA expression were analyzed.
  
  
  RESULTS
  The data obtained suggest that U6 promoter-enhanced GlnUAG tRNAs have higher suppression efficacies than multimers of the same suppressor tRNA without a U6 promoter. Proteomic analysis of cells constitutively expressing the GlnUAG suppressor tRNA indicates that stable expression of NSTs may not lead to significant read through of normal cellular proteins.
  
  
  CONCLUSIONS
  Because most tRNAs have cell-specific differential expression, this technique will enable the expression of different kinds of suppressor tRNAs in various cell types at high, functionally relevant levels. The techniques developed in the present study may contribute to the further development of suppressor tRNA-mediated gene therapy.
authors.affiliation: Genetics, Cellular and Developmental Biology, Iowa State University, Ames, IA, USA.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Koukuntla
authors.forename: William J
authors.initials: WJ
authors.lastname: Ramsey
authors.forename: Won-Bin
authors.initials: WB
authors.lastname: Young
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Link
mesh_terms: D000926:Anticodon / Q000737:chemistry / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D018389:Codon, Nonsense; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D016153:Genes, Suppressor; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D011401:Promoter Regions, Genetic; D040901:Proteomics; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000926:Anticodon; D018389:Codon, Nonsense; D005973:Glutamine; D012343:RNA, Transfer