A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system

被引:42
作者
Igoucheva, O
Peritz, AE
Levy, D
Yoon, K [1 ]
机构
[1] Thomas Jefferson Univ, Dept Dermatol & Cutaneous Biol, Philadelphia, PA 19107 USA
[2] Thomas Jefferson Univ, Dept Biochem & Mol Pharmacol, Jefferson Inst Mol Med, Philadelphia, PA 19107 USA
[3] Thomas Jefferson Univ, Jefferson Med Coll, Philadelphia, PA 19107 USA
关键词
site-specific gene conversion; point mutation; homologous recombination; gene therapy;
D O I
10.1038/sj.gt.3301042
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
The variability in gene conversion frequency by an RNA-DNA oligonucleotide (RDO) prompted us to develop a system as a means of measuring the conversion frequency rapidly and reproducibly. A shuffle vector was constructed to measure the frequency of targeted gene correction by RDO of the E. coli beta-galactosidase gene containing a single point mutation (G --> A), that resulted in inactivation of enzymatic activity. An RDO corrected the point mutation and restored the enzymatic activity, approximately 1%, determined by a histochemical staining in mammalian cells and by a color selection (blue or white) of bacteria transformed with Hirt DNA. In addition, we established an in vitro system capable of gene correction using nuclear extracts. CHO-KI nuclear extracts corrected the point mutation approximately 0.1%, determined by bacterial transformation. Using the in vitro reaction, frequency of gene conversion in different cell types was measured. The embryonic fibroblasts from p53-/- mouse showed higher gene correction than that of the isogenic p53+/+ cells. Nuclear extracts from DT40 cells, which have a higher homologous recombination rate than any other mammalian cells exhibited 0.1-0.6% of gene correction. These results indicated that recombination may be rate-limiting in gene conversion by RDO in cells with competent mismatch repair activities. Utilizing transfection and in vitro reaction, we demonstrated that such a shuttle system might be useful in comparing the frequency of targeting among different cell types and to investigate the mechanism of gene conversion by RDO.
引用
收藏
页码:1960 / 1971
页数:12
相关论文
共 42 条
[1]   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
[2]   INCREASED RATIO OF TARGETED TO RANDOM INTEGRATION AFTER TRANSFECTION OF CHICKEN B-CELL LINES [J].
BUERSTEDDE, JM ;
TAKEDA, S .
CELL, 1991, 67 (01) :179-188
[3]   HIGH MUTATION FREQUENCY IN DNA TRANSFECTED INTO MAMMALIAN-CELLS [J].
CALOS, MP ;
LEBKOWSKI, JS ;
BOTCHAN, MR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1983, 80 (10) :3015-3019
[4]   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
[5]   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
[6]   A SET OF LACZ MUTATIONS IN ESCHERICHIA-COLI THAT ALLOW RAPID DETECTION OF EACH OF THE 6 BASE SUBSTITUTIONS [J].
CUPPLES, CG ;
MILLER, JH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (14) :5345-5349
[7]  
CUPPLES CG, 1988, GENETICS, V120, P637
[8]   Processing of three different types of DNA damage in cell lines of a cutaneous squamous cell carcinoma progression model [J].
Diem, C ;
Runger, TM .
CARCINOGENESIS, 1997, 18 (04) :657-662
[9]   ACCURATE TRANSCRIPTION INITIATION BY RNA POLYMERASE-II IN A SOLUBLE EXTRACT FROM ISOLATED MAMMALIAN NUCLEI [J].
DIGNAM, JD ;
LEBOVITZ, RM ;
ROEDER, RG .
NUCLEIC ACIDS RESEARCH, 1983, 11 (05) :1475-1489
[10]   Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role in fidelity control of homologous recombination [J].
Dudenhöffer, C ;
Rohaly, G ;
Will, K ;
Deppert, W ;
Wiesmüller, L .
MOLECULAR AND CELLULAR BIOLOGY, 1998, 18 (09) :5332-5342