Development of a dual-luciferase fusion gene as a sensitive marker for site-directed DNA repair strategies

Department of Microbiology, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
The Journal of Gene Medicine (Impact Factor: 2.47). 08/2003; 5(8):723-32. DOI: 10.1002/jgm.386
Source: PubMed


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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    • "The luciferase gene encodes a protein that produces light through an enzymatic reaction and is particularly attractive because it is sensitive and can be easily quantified. Repairing a mutated gene instead of inducing a mutation is preferable because it is easier to measure an increase in function above background than to detect a small decrease in a high level of activity [38]. The peGFPLucMut plasmid (Figure 1A) has a premature stop codon generated by a single nucleotide change in the open reading frame, upstream of the luciferase enzyme catalytic site. "
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