CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single-step procedure
CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single-step procedure
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DOI：
10.1038/gt.2010.89
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发表时间：
2010-11-01
期刊：
影响因子：
5.1
通讯作者：
Ascenzioni, F.
中科院分区：
文献类型：
--
作者：
Auriche, C.;Di Domenico, E. G.;Ascenzioni, F.
我们组装了两个BAC载体，其中包含一个跨越整个CFTR基因座的单一片段，包括上游和下游区域。这两个载体在上游区域的大小不同，并在大肠杆菌中回收，并准备了完整的BAC DNA用于结构和功能分析。序列分析使插入片段的精确映射成为可能。我们发现CFTR基因是野生型，是高加索人群中最常见的单倍型，通过以下多态性鉴定：(GATT)7位于内含子6a；(TG)(11)T-7位于内含子8；V470位于第470位。用RT-PCR、实时定量聚合酶链式反应、免疫印迹、间接免疫荧光和电生理等方法分析了CFTR在模型细胞中的表达和活性，表明CFTRCl通道具有活性。最后，支持CFTR作为铜绿假单胞菌受体的假设，我们证明了表达CFTR的细胞比不表达CFTR的亲本细胞内化了更多的细菌。总体而言，这些数据表明，BAC载体包含一个功能性CFTR片段，并具有独特的功能，包括从单个片段衍生，可获得详细的基因组图谱，以及使用标准提取程序制备BAC DNA的可能性。基因治疗(2010年)17,1341-1354；DOI：10.1038/gt.2010.89；2010年6月10日在线发布
We have assembled two BAC vectors containing a single fragment spanning the entire CFTR locus and including the upstream and downstream regions. The two vectors differ in size of the upstream region, and were recovered in Escherichia coli, with intact BAC DNAs prepared for structural and functional analyses. Sequence analysis allowed precise mapping of the inserts. We show that the CFTR gene was wild type and is categorized as the most frequent haplotype in Caucasian populations, identified by the following polymorphisms: (GATT) 7 in intron 6a; (TG)(11)T-7 in intron 8; V470 at position 470. CFTR expression and activity were analyzed in model cells by RT-PCR, quantitative real-time PCR, western blotting, indirect immunofluorescence and electrophysiological methods, which show the presence of an active CFTR Cl channel. Finally, and supporting the hypothesis that CFTR functions as a receptor for Pseudomonas aeruginosa, we show that CFTR-expressing cells internalized more bacteria than parental cells that do not express CFTR. Overall, these data demonstrate that the BAC vectors contain a functional CFTR fragment and have unique features, including derivation from a single fragment, availability of a detailed genomic map and the possibility to use standard extraction procedures for BAC DNA preparations. Gene Therapy (2010) 17, 1341-1354; doi:10.1038/gt.2010.89; published online 10 June 2010