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

被引:139
作者
Igoucheva, O
Alexeev, V
Yoon, K
机构
[1] Thomas Jefferson Univ, Jefferson Inst Mol Med, Dept Dermatol & Cutaneous Biol, Philadelphia, PA 19107 USA
[2] Thomas Jefferson Univ, Jefferson Inst Mol Med, Dept Mol Pharmacol & Biochem, Philadelphia, PA 19107 USA
[3] Jefferson Med Coll, Philadelphia, PA 19107 USA
关键词
site-specific gene correction; mutant beta-galactosidase; short single-stranded oligonucleotide; gene repair;
D O I
10.1038/sj.gt.3301414
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 beta -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 chromosomal gene corrections were highly dependent on the ODN length and polarity. ODN with a homology of 45 nucleotides showed the highest frequency and ODN with antisense orientation showed a 1000-fold higher frequency than sense orientation, indicating a possible influence of transcription on gene correction. Deoxyoligonucleotides showed a higher frequency of gene correction than ribo-oligonucleotides of the identical sequence. These results show that a relatively short ODN can make a sequence-specific change in the target sequence in mammalian cells, at a similar frequency as the chimeric RNA-DNA oligonucleotide.
引用
收藏
页码:391 / 399
页数:9
相关论文
共 42 条
[1]   Gene correction by RNA-DNA oligonucleotides [J].
Alexeev, V ;
Yoon, K .
PIGMENT CELL RESEARCH, 2000, 13 (02) :72-79
[2]   Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotide [J].
Alexeev, V ;
Yoon, K .
NATURE BIOTECHNOLOGY, 1998, 16 (13) :1343-1346
[3]   In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide [J].
Bartlett, RJ ;
Stockinger, S ;
Denis, MM ;
Bartlett, WT ;
Inverardi, L ;
Le, TT ;
Man, NT ;
Morris, GE ;
Bogan, DJ ;
Metcalf-Bogan, J ;
Kornegay, JN .
NATURE BIOTECHNOLOGY, 2000, 18 (06) :615-622
[4]   A tool for functional plant genomics:: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations [J].
Beetham, PR ;
Kipp, PB ;
Sawycky, XL ;
Arntzen, CJ ;
May, GD .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (15) :8774-8778
[5]   DNA-REPAIR - ENGAGEMENT WITH TRANSCRIPTION [J].
BOOTSMA, D ;
HOEIJMAKERS, JHJ .
NATURE, 1993, 363 (6425) :114-115
[6]  
CAMPBELL C R, 1989, New Biologist, V1, P223
[7]   Targeted correction of an episomal gene in mammalian cells by a short DNA fragment tethered to a triplex-forming oligonucleotide [J].
Chan, PP ;
Lin, M ;
Faruqi, AF ;
Powell, J ;
Seidman, MM ;
Glazer, PM .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (17) :11541-11548
[8]   Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract [J].
Cole-Strauss, A ;
Gamper, H ;
Holloman, WK ;
Muñoz, M ;
Cheng, N ;
Kmiec, EB .
NUCLEIC ACIDS RESEARCH, 1999, 27 (05) :1323-1330
[9]   Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide [J].
ColeStrauss, A ;
Yoon, KG ;
Xiang, YF ;
Byrne, BC ;
Rice, MC ;
Gryn, J ;
Holloman, WK ;
Kmiec, EB .
SCIENCE, 1996, 273 (5280) :1386-1389
[10]   Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides [J].
Culver, KW ;
Hsieh, WT ;
Huyen, Y ;
Chen, V ;
Liu, JL ;
Khripine, Y ;
Khorlin, A .
NATURE BIOTECHNOLOGY, 1999, 17 (10) :989-993