PILOT STUDY--DEVELOPMENT OF TRANSGENIC CF MICE WITH SPECIFIC MUTATIONS

直到最近，还没有已知的囊性纤维化的动物模型。 随着小鼠囊性纤维化基因的分离， 开发囊性纤维化的小鼠模型。三种小鼠模型已经被 报道，并通过破坏CFTR基因，通过同源 重组而其中两只动物是基因“敲除”， CF的独特胃肠道表现，另一只动物具有 重复的同源序列彼此相邻插入， CF疾病症状较轻。两种动物之间的差异 表明CF表型将根据CFTR突变而变化。在 然而，目前没有动物具有特异性CFTR突变， 类似于人类。 因为上面描述的动物不 代表自然发生的CFTR突变， 从他们身上可以学到什么关于一个给定的 CF病理和确定的CFTR突变。我们建议使用一种新的基因 开发具有不同CFTR，外显子10的CF小鼠的靶向方案 突变。小片段同源重组（SFHR）策略 将在小鼠129中引入同源DNA的小片段（392 bp） 胚胎干细胞（ES细胞）。片段将包含特定的外显子10 突变以及新的限制性内切酶切割位点， 野生型（WT）外显子10。同源重组将通过 基因组基因座的聚合酶链反应（PCR）扩增 跨越同源序列（435 bp）和随后的限制性内切酶切 PCR产物的酶消化。富集的重组群体 将CF杂合子ES细胞经PCR筛选后分离出ES细胞 以克隆密度铺板的细胞集落。确定为主要 CF杂合子将扩大用于基因组DNA分析和测量 野生型：CF杂合子比率。殖民地有一个大于 70%比例的CF杂合子细胞将用于囊胚 注射嵌合动物将通过PCR筛选，然后评估 生殖系传播 将饲养杂合子小鼠，CF纯合子小鼠 将对动物进行表型和基因型表征。 PCR 扩增和基因组Southern杂交将用于 验证动物的突变状态。CF的表型状态 小鼠将通过疾病的形态学评估来确定 病理是各种器官（例如，肠梗阻，气道 分泌）和上皮的Cl离子转运特性。 机关 将通过光学电子显微镜和分离的上皮细胞进行分析。 将测定细胞的36Cl的cAMP依赖性流出。

Until recently there have been no known animal models for cystic fibrosis. With isolation of the mouse cystic fibrosis gene it became possible to develop mouse models of cystic fibrosis. Three mouse models have been reported and were produced by disrupting the CFTR gene through homologous recombination. While two of the animals are gene "knockouts" with distinctive gastrointestinal manifestations of CF, the other animal has duplicate homologous sequences inserted adjacent to one another and has milder CF disease symptoms. The variation between the two types of animals indicates that CF phenotype will vary depending on the CFTR mutation. At present there are, however, no animals with specific CFTR mutations analogous to those in humans. Because the animals described above do not represent naturally occurring CFTR mutations, there will be limitations on what can be learned from them regarding the relationship between a given CF pathology and a defined CFTR mutation. We propose to use a novel gene targeting protocol to develop CF mice with distinct CFTR,exon 10 mutations. The small fragment homologous recombination (SFHR) strategy will introduce small fragments (392 bp) of homologous DNA in mouse 129 embryonic stem (ES) cells. The fragments will contain specific exon 10 mutations as well as new restriction enzyme cleavage sites not found in wild type (wt) exon 10. Homologous recombination will be assessed by polymerase chain reaction (PCR) amplification of the genomic locus spanning the homologous sequences (435 bp) and subsequent restriction enzyme digestion of the PCR products. Enriched populations of recombinant CF heterozygote ES cells will be obtained after PCR screening isolated ES cell colonies plated at clonal density. Clones identified as predominantly CF heterozygotes will be expanded for genomic DNA analysis and measurement of the wild-type:CF heterozygte ratio. Colonies that have a greater than 70% proportion of CF heterozygote cells will be used for blastocyst injection. Chimeric animals will be screened by PCR and then assessed for germline transmission. Heterozygote mice will be bred and CF homozygote animals will be phenotypically and genotypically characterized. PCR amplification and genomic Southern hybridization will be employed to verify the mutation status of the animals. The phenotypic status of the CF mice will be determined by morphological assessment of the disease pathology is various organs (e.g., intestinal obstruction, airway' secretion) and the Cl ion transport properties of the epithelium. Organs will be analyzed by light electron microscopy and isolated epithelial cells will be assayed for CAMP-dependent efflux of 36Cl.