Mogayzel P J, Henning K A, Bittner M L, Novotny E A, Schwiebert E M, Guggino W B, Jiang Y, Rosenfeld M A
Laboratories of Gene Transfer, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hum Mol Genet. 1997 Jan;6(1):59-68. doi: 10.1093/hmg/6.1.59.
The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH). Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact. Northern analysis showed full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR. This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.
囊性纤维化跨膜传导调节因子基因（CFTR）编码一种跨膜蛋白（CFTR），其部分功能是作为一种环磷酸腺苷（cAMP）调节的氯离子通道。CFTR的表达受时间和细胞特异性的调控，但其调控机制尚不清楚。成功地将完整的230 kb人类CFTR及其相邻序列导入哺乳动物细胞，有助于深入了解CFTR的调控机制。为此，我们将两种不同的含CFTR的酵母人工染色体（YACs）（320 kb和620 kb）导入中国仓鼠卵巢-K1（CHO）细胞。通过PCR鉴定出含有人CFTR的克隆细胞系，并对每个YAC的一个克隆进行了遗传和功能分析。通过荧光原位杂交（FISH）证明了含有人CFTR的YACs在每个细胞系的独特位点整合到CHO基因组中。Southern印迹分析表明，每个CHO细胞基因组中大约整合了一个拷贝的人CFTR。纤维-FISH和限制性分析表明CFTR大体上保持完整。Northern分析显示有全长的人CFTR mRNA。免疫沉淀后用蛋白激酶磷酸化证明有成熟的、糖基化的CFTR。最后，对cAMP的氯离子分泌反应表明了人CFTR的功能特性。本研究提供了几个新的结果，包括：（i）功能性人CFTR可以从这些YACs中表达；（ii）CHO细胞是表达人CFTR的允许环境；（iii）鉴于缺乏内源性CFTR产生，CHO细胞中人CFTR的表达水平出乎意料地高；（iv）纤维-FISH提示CFTR完整性与功能基因表达相关。这些YACs及其衍生的细胞系应该是研究CFTR表达的有用工具。
