Development of a dual-luciferase fusion gene as a sensitive marker for site-directed DNA repair strategies
ABSTRACT Several novel techniques have been developed recently for the site-specific repair of DNA as an approach to gene therapy. Correction efficiencies as high as 40% have been reported, well within the range of therapeutic impact for a number of genetic diseases. Unfortunately, many of the model systems in which these methods have been employed typically target genes that are not well suited for analyzing the various techniques.
To address this, we have constructed and characterized a dual-luciferase fusion gene as a sensitive marker for optimizing repair strategies. The genes encoding two distinct luciferase proteins were fused so that expression of one luciferase necessitated expression of the other. Engineering a stop codon in the downstream luciferase gene created an ideal tool to study the efficiency of various site-directed DNA repair techniques as one luciferase can act as an internal control while the other is targeted for correction.
Fusing two luciferase genes resulted in a single protein that produces two bioluminescent activities in a constant ratio. The utility of this system as a target for site-directed DNA repair research was demonstrated using two of the recently developed gene repair techniques, small fragment homologous replacement and oligonucleotide-mediated repair, to mediate correction and by the ability to detect repair efficiencies of less than 5 x 10(-6) (<1 event in 200000).
The ability to rapidly and accurately quantify the amount of correction using the dual-luciferase fusion system will allow the comparison and evaluation of the many factors involved in successful gene repair and lead to the optimization of these techniques, both in cell culture and in whole animals.
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ABSTRACT: The site-specific insertion of an unnatural amino acid into proteins in vivo via nonsense suppression has resulted in major advances in recent years. The ability to incorporate two different unnatural amino acids in vivo would greatly increase the scope and impact of unnatural amino acid mutagenesis. Here, we show the concomitant suppression of an amber and an ochre codon in a single mRNA in mammalian cells by importing a mixture of aminoacylated amber and ochre suppressor tRNAs. This result provides a possible approach to site-specific insertion of two different unnatural amino acids into any protein of interest in mammalian cells. To our knowledge, this result also represents the only demonstration of concomitant suppression of two different termination codons in a single gene in vivo.Chemistry & Biology 12/2003; 10(11):1095-102. DOI:10.1016/j.chembiol.2003.10.013 · 6.59 Impact Factor
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ABSTRACT: We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.Nucleic Acids Research 02/2004; 32(21):6200-11. DOI:10.1093/nar/gkh959 · 9.11 Impact Factor
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ABSTRACT: Defects in the dystrophin gene cause the severe degenerative muscle disorder, Duchenne muscular dystrophy (DMD). Among the gene therapy approaches to DMD under investigation, a gene editing approach using oligonucleotide vectors has yielded encouraging results. Here, we extend our studies of gene editing with self-pairing, chimeric RNA/DNA oligonucleotides (RDOs) to the use of oligodeoxynucleotides (ODNs) to correct point mutations in the dystrophin gene. The ODN vectors offer many advantages over the RDO vectors, and we compare the targeting efficiencies in the mdx(5cv) mouse model of DMD. We found that ODNs targeted to either the transcribed or the non-transcribed strand of the dystrophin gene were capable of inducing gene repair, with efficiencies comparable to that seen with RDO vectors. Oligonucleotide-mediated repair was demonstrated at the genomic, mRNA and protein levels in muscle cells both in vitro and in vivo, and the correction was stable over time. Interestingly, there was a strand bias observed with the ODNs, with more efficient correction of the non-transcribed strand even though the dystrophin gene is not transcribed in proliferating myoblasts. This finding demonstrates that strand bias of ODN-mediated gene repair is likely to be due to the specific sequence of the target gene in addition to any effects of transcription. A better understanding of how the efficiency of gene editing relates to the target sequence will offer the opportunity for rational oligonucleotide design for further development of this elegant approach to gene therapy for DMD and other genetic diseases.Human Molecular Genetics 02/2005; 14(2):221-33. DOI:10.1093/hmg/ddi020 · 6.68 Impact Factor