文章基本信息

标题：Reversal of cystic fibrosis phenotype in a cultured Δ508 cystic fibrosis transmembrane conductance regulator cell line by oligonucleotide insertion
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作者：Paul C. Zamecnik ; Malay K. Raychowdhury ; David R. Tabatadze 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：21
页码：8150-8155
DOI：10.1073/pnas.0401933101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Cystic fibrosis (CF) is a lethal genetic disorder that is due to mutations in the gene encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. A three-nucleotide base deletion (TTT), encoding phenylalanine in position 508 of the translatable CFTR sequence (accompanied by a C to T replacement immediately 5' to the deletion), accounts for {approx}75% of cases of the disease. In the present study, an oligonucleotide complex (CF4-CF6, 2'-0-methyl RNA-unmodified RNA oligonucleotide duplex, respectively) was used to restore CFTR function by insertion of missing bases in {Delta}508 CFTR mRNA from a cultured ({Delta}508) cell line. cAMP-activated whole-cell currents and Cl- transport were detected in CF4-CF6-treated, but not control {Delta}508, cells by patch-clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence (SPQ) quenching analyses, respectively. Further, the nucleotide addition in the deleted region of {Delta}508 CFTR was determined after amplification by RT-PCR. Insertion of UGU and replacement of U by C immediately 5' to the deletion site in {Delta}508 mRNA appear to have taken place, with phenotypic but not genotypic reversion in tissue culture of treated cells. The mechanism of insertion of nucleotides has yet to be determined.
关键词：tissue culture ; functional restoration ; mRNA repair