Targeted gene correction by small single-stranded oligonucleotides in mammalian cells

Abstract

We demonstrate that relatively short single-stranded oligodeoxynucleotides, 25-61 bases homologous to the target sequence except for a single mismatch to the targeted base, are capable of correcting a single point mutation (G to A) in the mutant ␤-galactosidase gene, in nuclear extracts, episome, and chromosome of mammalian cells, with correction rates of approximately 0.05%, 1% and 0.1%, respectively. Surprisingly, these short single-stranded oligonucleotides (ODN) showed a similar gene correction frequency to chimeric RNA-DNA oligonucleotide, measured using the same system. The in vitro gene correction induced by ODN in nuclear extracts was not dependent on the length or polarity of the oligonucleotide. In contrast, the episomal and

A comparison of gene repair strategies in cell culture using a lacZ reporter system

“…In particular, while a 0.52 kb PCR-generated fragment gave the highest level of episomal plasmid repair, it was the single-stranded antisense oligonucleotides of 35 or 80 bases that Comparison of gene repair strategies HD Nickerson and WH Colledge gave the highest frequencies of repair at a chromosomal locus. Our data agree with previous reports that an antisense oligonucleotide increases the frequency of chromosomal gene repair above that obtained with a sense oligonucleotide, although we recorded a difference of only 5 to 10-fold, which is considerably lower than the 1000-fold reported Igoucheva et al 7 This difference might be caused by a transcriptional effect, as it is possible a sense oligonucleotide to be displaced from the transcribed strand of DNA by the RNA polymerase II complex during transcription. 20 More stable binding of the antisense oligonucleotide to the nontranscribed strand might account for its relatively higher frequencies of gene repair.…”

“…This mutation was previously used to evaluate the efficacy of RNA:DNA and single-stranded DNA oligonucleotides for gene repair. 7,15 The mutated plasmid, pCMVM1bgeo, bears a b-galactosidase -neomycin resistance fusion gene, bgeo, which produces a fusion protein product, bGEO. Although the b-galactosidase part of this protein is not functional unless gene repair has occurred, the protein can be detected by Western blotting, and the portion of the protein conferring neomycin resistance is still functional and was used to generate stable cell lines.…”

“…3 In addition, early reports suggested that single-stranded oligonucleotides of only 40 bases were sufficient to achieve gene targeting on a plasmid. 4 More recently, somatic cell gene targeting has been achieved in vitro with RNA:DNA chimeric oligonucleotides [5][6][7][8] double-and single-stranded PCR-generated fragments 2,3,9 and single-stranded oligonucleotides. Gene repair has also been reported in vivo in a variety of tissues including the liver, 10 , lung, 11 muscle, 12 and skin, 13 using a variety of fragments or oligonucleotides.…”

Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells

“…We developed an experimental system utilizing relatively short single-stranded oligodeoxynucleotides (ODNs) that allow direct site-specific correction of mutations in endogenous genes. 31 Several characteristics of the ODNs make them an attractive tool for in vivo applications. The primary advantage of such an approach is the ability to make a permanent specific alteration into a gene of interest in a single step, without disturbing the genomic organization required for endogenous expression and regulation of the targeted gene.…”

“…The primary advantage of such an approach is the ability to make a permanent specific alteration into a gene of interest in a single step, without disturbing the genomic organization required for endogenous expression and regulation of the targeted gene. In the past 8 years, we have made significant progress in our understanding of the mechanism responsible for gene alteration by ODNs [32][33][34][35][36] and provided clear evidence that this approach can be used to introduce permanent site-specific changes into episomal and chromosomal DNA of somatic cells 31,37,38 and embryonic stem (ES) cells. 39,40 The goal of the present study was to assess the use of ODN-directed gene repair in MSCs.…”