Unmodified oligodeoxynucleotides require single-strandedness to induce targeted repair of a chromosomal EGFP gene.
BACKGROUND
A number of genetic defects in humans are due to point mutations in a single, often tightly regulated gene. Genetic treatment of such defects is preferably done by correcting only the altered base pair at the endogenous locus rather than by a gene replacement strategy involving viral vectors. Promisingly high repair rates have been achieved in some systems with the non-viral approach of transfecting chimeric RNA/DNA oligonucleotides (chimeraplasts). However, since this technique does not yet perform robustly, several parameters thought to be important in oligonucleotide-mediated gene repair were examined.


METHODS
A series of transgenic HEK-293 cell clones has been established harboring high or low copy numbers of a point-mutated 'enhanced green fluorescent protein' (EGFP) gene as the target. At the level of single living cells, repair efficiencies were measured by fluorescence-activated cell sorting (FACS) regarding topology (single-stranded, double-stranded), exonuclease protection (four phosphorothioate linkages at both ends), polarity (sense, antisense), and length (13mer, 19mer, 35mer, 69mer) of the oligonucleotide.


RESULTS
When targeting chromosomal loci, up to 0.2% corrected cells were obtained with single-stranded unmodified oligodeoxynucleotides, whereas a chimeraplast, its DNA analogue, and double-stranded DNA fragments were practically non-functional. Correction efficiencies correlated with target gene copy numbers. Modifying exonuclease resistance, polarity or length of single-stranded oligodeoxynucleotides did not enhance repair efficacy above the sub-percentage range.


CONCLUSIONS
Successful chromosomal reporter gene repair in HEK-293 cells required an oligodeoxynucleotide to be single-stranded. In concert with the gene copy number correlation, functional interaction between the repair molecule and the target site seems to be one bottleneck in targeted gene repair.
Radecke
Frank
F
Institut für Klinische Transfusionsmedizin und Immungenetik Ulm gGmbH, Abteilung Transfusionsmedizin, Universität Ulm, D-89081 Ulm, Germany.
Radecke
Sarah
S
Schwarz
Klaus
K
D002460:Cell Line; D002877:Chromosomes, Human / Q000235:genetics*; D004260:DNA Repair; D005434:Flow Cytometry; D018628:Gene Dosage; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D020320:Oligoribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D017354:Point Mutation
D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense; D020320:Oligoribonucleotides, Antisense; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins