Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors.
Determining the function of newly discovered genes is at the center of the evolving field of genomics. With the elucidation of the human DNA sequence, the importance of single base changes to gene function has become apparent. In some cases, nucleotide alteration accounts for inherited disorders, but in other cases, subtle, even conservative, base changes can influence the function of a gene and its product. To identify how critical genetic changes alter function, molecular tools such as synthetic vectors have been created to direct nucleotide exchange. Some of these vectors, including chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides, have shown promise in the specific alteration of a single base at an exact position within the gene. Here, we describe the activity of the synthetic vectors in a mammalian cell system. The episomal target contains a mutation in the neomycin resistance gene fused to a reporter ligand-binding domain. Correction of the mutated base enables translation of the normal fusion product. This protein can now bind a ligand, resulting in the expression of the fusion protein visualized by green fluorescence. Hence, the activity of any similar vector can be measured easily (and in real time) using confocal microscopy. The system provides the basis for examining the effectiveness of new targeting molecules for creating or repairing single base alterations. In addition, genes suspected of affecting the frequency of repair can be tested through their expression in cells harboring the mutated target plasmid. Once the frequency of exchange in cells is established, the use of these vectors will become commonplace in a process designed to generate specific single base changes in genes involved in signal transduction. Such changes should help define functional domains within these proteins.
Parekh-Olmedo
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The Department of Biological Sciences and the Delaware Biotechnology Institute, University of Delaware, Newark, DE 19716, USA.
Czymmek
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Kmiec
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EB
D000818:Animals; D004247:DNA / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D018390:Gene Targeting / Q000295:instrumentation / Q000379:methods*; D015316:Genetic Therapy / Q000295:instrumentation / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D051381:Rats; D011911:Rats, Gunn; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D008565:Membrane Proteins; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA