Gene targeting of CFTR DNA in CF epithelial cells.
Gene targeting of CFTR DNA in CF epithelial cells.
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DOI：
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发表时间：
1996-10
期刊：
影响因子：
5.1
通讯作者：
K. Kunzelmann;J. Legendre;D. Knoell;L. C. Escobar;Z. Xu;D. Gruenert
中科院分区：
文献类型：
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作者：
K. Kunzelmann;J. Legendre;D. Knoell;L. C. Escobar;Z. Xu;D. Gruenert
囊性纤维化 (CF) 基因治疗的目标是用野生型 (wt) DNA 序列纠正突变的 CF 跨膜电导调节 (CFTR) 基因，以恢复正常的 CFTR 蛋白和功能。 wtCFTR cDNA 表达载体的实验表明，与 CF 相关的 Cl 离子转运表型可以被校正为类似于正常细胞中的表型。基于 cDNA 的基因治疗策略的替代方案是通过用 wt 同源物进行靶向替换来纠正内源突变序列。为了测试这种策略是否可行，使用小片段同源替换（SFHR）策略来替换人上皮细胞中的特定基因组序列。将基因组 wtCFTR DNA 的小片段转染至转化的 CF 上皮细胞中。在适当的基因组位点被外源 CFTR DNA 取代及其作为 mRNA 的表达通过以下方式表明：（1）基因组 DNA 和 mRNA 衍生的 cDNA 的等位基因特异性聚合酶链反应（PCR）扩增； (2) PCR产物与等位基因特异性探针的杂交。此外，通过全细胞膜片钳测定CFTR蛋白的功能活性。 Southern 杂交和膜片钳分析表明，大约百分之一的 CF 细胞经历了同源替换事件，导致完整的 Cl 转运。
A goal of cystic fibrosis (CF) gene therapy is correction of the mutant CF transmembrane conductance regulator (CFTR) gene with wild-type (wt) DNA sequences to restore normal CFTR protein and function. Experiments with wtCFTR cDNA expression vectors have shown that the Cl ion transport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replacement with the wt homologue. To test whether such a strategy was feasible, a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fragments of genomic wtCFTR DNA were transfected into transformed CF epithelial cells. Replacement by exogenous CFTR DNA at the appropriate genomic locus and its expression as mRNA was indicated by: (1) allele-specific polymerase chain reaction (PCR) amplification of genomic DNA and mRNA-derived cDNA; and (2) hybridization of PCR products with allele-specific probes. In addition, the functional activity of CFTR protein was determined by whole cell patch clamp. Southern hybridization and patch clamp analyses suggested that approximately 1 in 100 CF cells underwent a homologous replacement event that resulted in intact Cl transport.