MOUSE MODELS FOR CYSTIC FIBROSIS

囊性纤维化小鼠模型

• 批准号: 2145220
• 负责人: ALLAN BRADLEY
• 金额: $ 35.58万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1996-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145220
• 关键词: cell line; clone cells; cystic fibrosis; disease /disorder model; electrophysiology; gene mutation; genetic strain; genetically modified animals; immunologic techniques; laboratory mouse; molecular cloning; mutant; phenotype; polymerase chain reaction; recombinant DNA; southern blotting; tissue mosaicism; western blottings

This proposal is based on two years of preliminary work and is a direct and specific response to a request for applications aimed at generating animal models of cystic fibrosis (CF). Specific goals of the project will be to prepare null mutations and to transmit these to the mouse germline. Depending on the phenotype obtained with a null mutation, additional missense mutations and hypomorphic mutations will be generated. The various mutations would be introduced on to different mouse backgrounds in order to examine the effect of genetic variation at other loci on the phenotype in the mouse. The CF mutations would be moved on to other mouse strains either by backcrossing or by preparing ES cell lines from different mouse strains. The phenotype of the mutant mice would be assessed clinically, pathologically, and physiologically. The investigators already have committed at least two years work towards the goal of obtaining mice with mutations in the CF gene. Numerous constructs have been prepared in the region corresponding to human exon 19 and in the region corresponding to human exons 2 and 3. Insertion and replacement vectors are being investigated. Recombinant clones are being screened using a mini-Southern blot protocol and using PCR to analyze pools of clones. Clones documented to represent homologous recombinants by Southern blotting will be injected into mouse blastocysts. Animals with a high percent chimerism will be bred in order to obtain germline transmission. Homozygous mutant animals will be assessed for survival, weight gain, susceptibility to infection, and other phenotypic features known to be associated with CF. Complete pathological studies will be performed on mutant animals. Any residual function of the CF gene will be assessed using reverse transcriptase-PCR, Western blotting, and immunostaining of tissue sections. Animals will be provided to collaborators for electrophysiological analysis. Immortalized cell lines will be developed from mutant and wild-type animals using a variety of strategies. As indicated by this request for applications, there is substantial reason to believe that mouse models for CF would be valuable in developing a better understanding of the pathophysiology, in assessing pharmacologic approaches, and in developing somatic gene therapy.

该建议基于两年的初步工作，是直接的 以及对旨在生成申请请求的具体回应 囊性纤维化的动物模型（CF）。 项目的具体目标 将准备无效突变并将其传输到小鼠 种系。 取决于用无效突变获得的表型 额外的错义突变和型肌差突变将是 生成。 各种突变将被引入不同 小鼠背景是为了检查遗传变异的影响 小鼠表型上的其他基因座。 CF突变将是 通过回击或准备ES，转移到其他鼠标菌株 来自不同小鼠菌株的细胞系。 突变体的表型 小鼠将在临床，病理和生理上进行评估。 调查人员已经承诺至少两年 在CF基因中获得突变的小鼠的目的。 很多的 构建体是在与人外显子相对应的区域中制备的 19，在对应于人类外显子2和3的地区。插入和 正在研究替换矢量。 重组克隆正在 使用迷你南方印迹方案进行筛选，并使用PCR进行分析 克隆池。 记录的克隆以表示同源重组 通过Southern印迹将注入小鼠胚泡。 动物 将有高百分比的嵌合为了获得种系 传播。 将评估纯合突变动物的生存， 体重增加，感染易感性和其他表型特征 已知与CF有关。 完整的病理研究将是 在突变动物上进行。 CF基因的任何残留功能都将 可以使用逆转录酶-PCR，Western blotting和 组织切片的免疫染色。 将提供动物 电生理分析的合作者。 永生的细胞系 将使用多种 策略。 如本申请请求所示，有 相信CF的鼠标模型将是有价值的实质理由 在更好地理解病理生理学时，评估 药理学方法，以及发展体细胞基因疗法。