TARGETED CFTR REPLACEMENT IN CF AIRWAY EPITHELIAL CELLS

大量细胞研究旨在阐明两者之间的关系 囊性纤维化跨膜电导调节剂与氯离子 运输使用全长的cDNA来产生一种功能蛋白 产品。因为这些调查涉及CFTR的过度表达， 他们不能评估CFTR水平在表达 一种给定的离子转运表型也不能评估体细胞的影响 遗传因素对Cf表型表达的影响概述的项目 这里将使用同源重组作为基因的替代 与cdna互补，将开发出具有特异性的细胞系。 Cf基因分型。这种理解CFTR函数的方法有几个方面 优点：1)将外源cftr序列置于 内源性cftr启动子并确保cftr在 给定细胞类型中的适当水平，2)它有助于分析 恒定遗传背景中的不同CFTR突变，即一个 突变会被同一细胞系中的另一个取代，以及3)它 使用的是一种正常表达CFTR的人体细胞系统。转型 本实验室培养的正常和囊性纤维化上皮细胞 将在这些研究中使用。初步的补充研究将使用 一株为Delta-F508突变纯合的CF细胞系。同源 含有基因组CFTR序列的重组载体将被 通过电穿孔进入细胞。即将到来的基因组CFTR 序列将覆盖CFTR的一个区域，包括外显子10和 5‘和3’内含子序列侧翼。靶向基因的两种策略 将使用更换。一种战略将采用替代载体。 将构建替换载体，以使neo(R)基因 包含在侧翼内含子序列中。HSV-tk基因将是 与同源区域相邻，因此将被消除 在同源重组过程中。同源重组体将被 由正/负选择(PNS)方案选择，该方案丰富了 同源重组子。PNS依赖于G418的积极选择 对存在疱疹的耐药性(NEO(R))和阴性选择 单纯病毒胸苷激酶(HSV-tk)基因。另一种方法将 使用插入载体并依靠染色体内重组 消除选择标记和重复的基因组序列。CF型电池 具有特定CF型的品系将由同源 用一个CF等位基因替换另一个。同源重组体将 通过外流和外流来表征其氯离子传输特性 膜片钳分析测定~(36)CI外流及全细胞和单细胞 分别为通道氯电流。此外，重组细胞将 通过检测CFTRmRNA和蛋白的表达来确定其作用 一个特定的cftr突变对cftr表达水平的影响以及 相关表型。

Numerous cellular studies designed to elucidate the relationship between the cystic fibrosis transmembrane conductance regulator (CFTR) and Cl ion transport employ a full-length cDNA that produces a functional protein product. Because these investigations involve overexpression of CFTR, they cannot evaluate the role that CFTR levels play in the expression of a given ion transport phenotype nor can they assess the effect of somatic genetic factors on expression of the CF phenotype. The project outlined here will use homologous recombination as an alternative to gene complementation with cDNA and will develop cell lines that have specific CF genotypes. This approach to understanding CFTR function has several advantages: 1) it places exogenous CFTR sequences under the regulation of the endogenous CFTR promoter and ensures that CFTR is expressed at appropriate levels in a given cell type, 2) it facilitates analysis of different CFTR mutations in a constant genetic background, i.e., one mutation will be replaced by another in the same cell line, and 3) it uses a human cell system that normally expresses CFTR. Transformed normal and cystic fibrosis epithelial cells developed in this laboratory will be used in these studies. Initial complementation studies will use a CF cell line, homozygous for the delta-F508 mutation. Homologous recombination vectors containing genomic CFTR sequences will be introduced into the cells by electroporation. The incoming genomic CFTR sequences will cover a region of CFTR that includes exon 10 and the flanking 5' and 3' intron sequences. Two strategies for targeted gene replacement will be used. One strategy will employ replacement vectors. The replacement vector, will be constructed such that the neo(r) gene is contained within flanking intron sequences. The HSV-tk gene will be adjacent to the region of homology and will therefore be eliminated during homologous recombination. Homologous recombinants will be selected by a positive/negative selection (PNS) scheme that enriches for homologous recombinants. PNS relies on positive selection for G418 resistance (neo(r)) and negative selection for the presence of the herpes simplex virus thymidine kinase (HSV-tk) gene. The other approach will use insertional vectors and rely on intrachromosomal recombination to eliminate the selection markers and duplicate genomic sequences. CF cell lines with a specific CF genotype will be generated by homologous replacement of one CF allele with another. Homologous recombinants will be characterized for their Cl ion transport properties by efflux and patch clamp analyses measuring 36CI efflux and whole-cell and single channel Cl currents, respectively. In addition, recombinant cells will be assayed for expression of CFTR mRNA and protein to determine the role that a specific CFTR mutation has on the level of CFTR expression and the associated phenotype.