Isolation, characterization and targeted disruption of mouse Ppia:: Cyclophilin A is not essential for mammalian cell viability
Isolation, characterization and targeted disruption of mouse Ppia:: Cyclophilin A is not essential for mammalian cell viability
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DOI：
10.1006/geno.2000.6295
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发表时间：
2000-09-01
期刊：
影响因子：
4.4
通讯作者：
Luban, J
中科院分区：
文献类型：
--
作者：
Colgan, J;Asmal, M;Luban, J
亲环素（CyPs）是在从原核生物到人类的生物体中发现的蛋白质家族。这些分子在体外表现出肽基脯氨酰异构酶活性，表明它们影响细胞中蛋白质的coni形成。CyPs还以不同的亲和力与免疫抑制药物环孢菌素A（CsA）结合，CsA是一种临床上用于预防同种异体移植排斥反应的化合物。该家族的创始成员亲环素A（CyPA）是一种丰富的、普遍表达的蛋白质，其功能未知，以纳摩尔亲和力与CsA结合。在这里，我们描述了小鼠Ppia（mPpia），编码CyPA的基因的分离和表征。使用区分表达的基因与小鼠基因组中存在的多个假基因的PCR筛选分离PPIA。mPpia由5个外显子和4个内含子组成，跨度约4.5 kb，定位于11号染色体着丝粒附近。mPpia近端启动子区的369 bp片段的序列分析显示存在TATA盒和被几种转录调节因子识别的位点，包括Spl、AP-2、加塔因子、c-Myb和NF-IL-6。该区域足以驱动转染细胞中的高水平报告基因表达。通过基因靶向在鼠胚胎干（ES）细胞中破坏Ppia的两个拷贝。Ppia（-/-）ES细胞在体外正常生长并分化为造血前体细胞，表明CyPA对哺乳动物细胞活力不是必需的。(C)北京大学出版社.
Cyclophilins (CyPs) are a family of proteins found in organisms ranging from prokaryotes to humans. These molecules exhibit peptidyl-prolyl isomerase activity in vitro, suggesting that they influence the coni formation of proteins in cells. CyPs also bind with varying affinities to the immunosuppressive drug cyclosporin A (CsA), a compound used clinically to prevent allograft rejection. The founding member of the family, cyclophilin A (CyPA), is an abundant, ubiquitously expressed protein of unknown function that binds with nanomolar affinity to CsA. Here, we describe the isolation and characterization of mouse Ppia (mPpia), the gene encoding CyPA. Ppia was isolated using a PCR screen that distinguishes the expressed gene from multiple pseudogenes present in the mouse genome. mPpia consists of 5 exons and 4 introns spanning roughly 4.5 kb and maps to chromosome 11 near the centromere. Sequence analysis of a 369-bp fragment from the proximal promoter region of: mPpia revealed the presence of a TATA box and sites recognized by several transcriptional regulators, including Spl, AP-2, GATA factors, c-Myb, and NF-IL-6. This region is sufficient to drive high-level reporter gene expression in transfected cells. Both copies of Ppia were disrupted in murine embryonic stem (ES) cells via gene targeting. Ppia(-/-) ES cells grow normally and differentiate into hematopoeitic precursor cells in vitro, indicating that CyPA is not essential for mam malian cell viability. (C) 2000 Academic Press.