Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting
Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting
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DOI：
10.1038/sj.gt.3301741
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发表时间：
2002-06-01
期刊：
影响因子：
5.1
通讯作者：
Gruenert, DC
中科院分区：
文献类型：
--
作者：
Bruscia, E;Sangiuolo, F;Gruenert, DC
囊性纤维化是高加索人群中最常见的遗传性疾病。约70%的CF染色体携带DeltaF 508突变，这是一种3-bp缺失，导致CF跨膜传导调节因子（CFTR）蛋白中氨基酸508处的苯丙氨酸丢失。通过小片段同源置换（SFHR）实现内源性CFTR的DeltaF 508基因座的直接修饰。用包含外显子10和侧翼内含子的野生型（WT）CFTR DNA的小片段（491-bp）转染对于DeltaF 508突变纯合的转化的人气道上皮细胞（CFBE 410（-））。DNA片段分别以6：1（+：-）的电荷比在脂质体-DNA复合物中。通过在96孔板中以类似于1个细胞/孔的有限稀释，将转染的细胞群体亚克隆。分离并分析单个菌落。通过放射性标记的非等位基因特异性和放射性标记的等位基因特异性PCR扩增以及基因组DNA指纹图谱对来自几个菌落的DNA进行表征。通过等位基因特异性PCR扩增在这些集落中的5个中检测到CFTR-WT等位基因，从而表明细胞系携带WT和DeltaF等位基因。DNA指纹分析证实，克隆是同基因的，并且来自亲本CFBE 41 o（-）细胞系。虽然通过等位基因特异性PCR检测到WT等位基因，但当通过非等位基因特异性PCR分析相同样品时，最初未检测到WT等位基因。混合野生型（16 HBE 14 o（-））和突变型（CFBE 41 o（-））细胞系的基因组DNA的灵敏度测定表明，等位基因特异性PCR比非等位基因特异性PCR灵敏至少25倍。这些结果表明，殖民地还不是克隆的，但仍含有未被修饰的亲本CFBE 41 o（-）细胞群。基于混合分析，校正细胞的比例似乎在总细胞群的1%至10%之间。CFTR mRNA的非等位基因特异性逆转录酶PCR（RT-PCR）分析表明，两个菌落同时表达WT和DeltaF 508 CFTR mRNA，而一个菌落似乎仅表达WT mRNA。通过来自CFTR外显子10的mRNA的3'末端引物延伸产物的序列分析证实了mRNA结果，表明该mRNA含有外显子10。此外，对引物延伸产物的调查表明，在表达的基因中没有随机插入片段。本研究证实了SFHR介导的DeltaF 508等位基因在DeltaF 508纯合子人气道上皮细胞中的多代修饰。所得细胞表达WT-CFTR mRNA，并且可以进一步亚克隆以分离细胞的等基因克隆群体。
Cystic fibrosis is the most common inherited disease in the Caucasian population. About 70% of all CF chromosomes carry the DeltaF508 mutation, a 3-bp deletion that results in the loss of a phenylalanine at amino acid 508 in the CF transmembrane conductance regulator (CFTR) protein. Direct modification of the DeltaF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR), Transformed human airway epithelial cells (CFBE41o(-)), homozygous for DeltaF508 mutation, were transfected with small fragments (491-bp) of wild-type (WT) CFTR DNA comprising exon 10 and the flanking introns. The DNA fragments were in a liposome-DNA complex at a charge ratio of 6:1 (+:-), respectively). The population of transfected cells was subcloned by limiting dilution at similar to1 cell/well in 96-well plates. Individual colonies were isolated and analyzed. The DNA from several colonies was characterized by radiolabeled, nonallele-specific and radiolabeled, allele-specific PCR amplification, as well as by genomic DNA fingerprinting. The CFTR-WT allele was detected in five of these colonies by allele-specific PCR amplification thus indicating that the cell lines carried both WT and DeltaF alleles. DNA fingerprint analysis confirmed that the colonies were isogenic and derived from the parental CFBE41o(-) cell line. Although, the WT allele was detected by allele-specific PCR, it was not detected initially when the same samples were analyzed by non allele-specific PCR. A sensitivity assay, mixing the genomic DNA of wild-type (16HBE14o(-)) and mutant (CFBE41o(-)) cell lines, indicated that the allele-specific PCR was at least 25-fold more sensitive than nonallele-specific PCR. These results suggest that the colony is not yet clonal, but still contains a population of parental, CFBE41o(-) cells that have not been modified. Based on the mixing analysis, the proportion of corrected cells appears to be between 1 and 10% of the total population. Nonallele-specific reverse transcriptase PCR (RT-PCR) analysis of the CFTR mRNA indicated that two of the colonies expressed both WT and DeltaF508 CFTR mRNA, while one colony appeared to express only the WT mRNA. The mRNA results were confirmed by sequence analysis of 3' end primer extension products from the mRNA of CFTR exon 10 showing that the mRNA containing exon 10. Furthermore, a survey of primer extension products indicated no random insertion of the fragment in an expressed gene. This study demonstrates SFHR-mediated modification of the DeltaF508 allele in DeltaF508 homozygote human airway epithelial cells over multiple generations. The resultant cells express WT-CFTR mRNA and can be subcloned further to isolate isogenic clonal populations of cells.