Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes.
Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes.
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由使用酵母人工染色体衍生的稳定中国仓鼠卵巢细胞系产生的功能性人类 CFTR。
DOI：
10.1093/hmg/6.1.59
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发表时间：
1997
影响因子：
3.5
通讯作者：
Rosenfeld,MA
中科院分区：
文献类型：
--
作者：
MogayzelJr,PJ;Henning,KA;Bittner,ML;Novotny,EA;Schwiebert,EM;Guggino,WB;Jiang,Y;Rosenfeld,MA
囊性自由化跨膜传导调节基因（cystic libiosis ti ansmembrane conductance regulator gene，CFTR）编码一种跨膜蛋白（transmembrane protein，CFTR），其部分功能是环磷酸腺苷（cyclicadenosine monophosphate，cAMP）调节的氯离子通道，其表达受时间和细胞特异性的调控，其调控机制尚不清楚。通过将完整的230 kb人CFTR及其邻近序列成功导入哺乳动物细胞，可以促进对CFTR调控的深入了解。为此，我们将两种不同的含CFTR的酵母人工染色体（YAC）（320和620 kb）导入中国仓鼠卵巢-K1（CHO）细胞。通过PCR鉴定含有人CFTR的克隆细胞系，并描述了含有每个YAC的一个克隆的遗传和功能分析。通过荧光原位杂交（FISH）证明了含有人CFTR的YAC在每个细胞系中的独特位点处整合到CHO基因组中。Southern印迹分析表明，每个CHO细胞基因组整合了一个拷贝的人CFTR。Fiber-FISH和酶切分析表明CFTR基本保持完整。北方分析显示全长人CFTRmRNA。免疫沉淀，然后与蛋白激酶磷酸化证明成熟的，糖基化的CFTR。最后，响应于cAMP的氯分泌表明了人CFTR的功能性质。该研究提供了几个新的结果，包括：（i）功能性人CFTR可以从这些YAC表达;（ii）CHO细胞是表达人CFTR的容许环境;（iii）考虑到缺乏内源性CFTR产生，CHO细胞中人CFTR表达的水平出乎意料地高;和（iv）Fiber-FISH提示CFTR完整性与功能性基因表达相关。这些YAC和细胞系衍生自他们应该是有用的工具，研究CFTR的表达。
The cystic libiosis ti ansmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel.CFTRexpression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight intoCFTRregulation could be facilitated by the successful introduction of the entire 230 kb humanCFTRand adjacent sequences into mammalian cells. To this end, we have introduced two differentCFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1(CHO) cells. Clonal cell lines containing humanCFTRwere identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the humanCFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescencein situhybridization (FISH). Southern blot analysis suggested that on the order of one copy of humanCFTRwas integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested thatCFTRremained grossly intact. Northern analysis showed full-length, humanCFTRmRNA. 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 humanCFTR;(iii) the level of humanCFTRexpression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH ofCFTRintegrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study ofCFTRexpression.