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U6 promoter enhanced GlnUAG suppressor tRNA has higher suppression efficacy and can be stably expressed in 293 cells.
MedLine Citation:
PMID:  23303531     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
BACKGROUND: Nearly one third of all human genetic diseases are due to 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 were adapted over the years to increase NST expression and efficacy, low suppression efficacies of NSTs and toxicity associated with stable expression of suppressor tRNAs have hampered development of NST mediated gene therapy. METHODS: We have employed the U6 promoter to enhance Gln-Amber suppressor tRNA (GlnUAG) expression and increase the PTC suppression in mammalian cells. In an attempt to study toxic effects of NSTs, a stable 293 cell line constitutively expressing a U6 promoter enhanced GlnUAG tRNA was established. To examine if 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: Our data suggest that U6 promoter enhanced Gln-Amber suppressor tRNAs (GlnUAG) 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: Since most tRNAs have cell-specific differential expression, this technique will enable expression of different kinds of suppressor tRNAs in various cell types at high, functionally relevant levels. The techniques developed here may contribute to the further development of suppressor tRNA mediated gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.
Authors:
Ramesh Koukuntla; William J Ramsey; Won-Bin Young; Charles J Link
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-10
Journal Detail:
Title:  The journal of gene medicine     Volume:  -     ISSN:  1521-2254     ISO Abbreviation:  J Gene Med     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-10     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9815764     Medline TA:  J Gene Med     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2013 John Wiley & Sons, Ltd.
Affiliation:
Genetics, Cellular and Developmental Biology, Iowa State University, Ames, Iowa.
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