REVERSE GENETICS OF CF LOCUS USING CHROMOSOME JUMPING

囊性纤维化（CF）是最常见的常染色体隐性遗传病之一 影响2500个白种新生儿中的1个。 这种疾病主要 影响肺部和胰腺，那里的损伤是由粘稠的 粘液积聚并导致阻塞和器官损伤。 的 目前的平均存活年龄约为25岁，死亡通常来自 呼吸衰竭 经过多年的工作，CF基因被克隆， 实验室与多伦多的儿童医院合作 在1989年，使用“反向遗传学”策略。 该基因编码1480 氨基酸膜蛋白，常见的突变是缺失 苯丙氨酸508. 我们现在提出一系列的研究，以进一步确定功能， CF基因的表达调控。 这将包括 通过多种方法鉴定导致CF的其他突变， 敏感技术，通过转染研究基因的功能 进入囊性纤维化细胞并评估缺陷的矫正， 基因的各个区域的位点特异性诱变， 所述抗体针对蛋白质产物的结构域，以便进一步 表征其表达，并研究转录调控 通过定义负责上皮特异性的启动子序列。 此外，我们最近发现了选择性剪接的证据， 可能具有重要功能后果的基因，以及 将对这一现象进行深入调查。 这些研究应 使我们更接近了解CF的正常功能 基因，并应奠定基础，发展有效的 治疗

Cystic fibrosis (CF) is one of the most common autosomal recessive diseases of man, affecting 1 in 2500 Caucasian newborns. The disease primarily affects the lungs and pancreas, where damage results from the thick sticky mucus which accumulates and leads to obstruction and organ damage. The current average survival is about age 25, with death usually coming from respiratory failure. After years of work, the CF gene was cloned by our laboratory in collaboration with the Hospital for Sick Children in Toronto in 1989, using the "reverse genetics" strategy. The gene encodes a 1480 amino acid membrane protein, and the common mutation is a deletion of phenylalanine 508. We now propose a series of studies to further define the function and regulation of expression of the CF gene. This will include the identification of additional mutations responsible for CF by a variety of sensitive techniques, a study of the function of the gene by transfection into cystic fibrosis cells and assessment of correction of the defect, site-specific mutagenesis of various regions of the gene, the generation of antibodies to domains of the protein product in order to further characterize its expression, and a study of the regulation of transcription by defining the promoter sequences responsible for epithelial specificity. Furthermore, we have recently identified evidence for alternative splicing of the gene which is likely to have important functional consequences, and this phenomenon will be intensively investigated. These studies should bring us much closer to an understanding of the normal function of the CF gene, and should lay the groundwork for the development of effective therapies.