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.
"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 . 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. "
[Show abstract][Hide abstract] ABSTRACT: Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome.
In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin.
Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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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