IDENTIFICATION OF AN ACTIVATOR HORMONE FOR A HEPATIC ORPHAN NUCLEAR RECEPTOR

FXR先前被鉴定为由FXR激活的核受体。 具有法尼基主链的分子。 我们继续在这方面开展工作， 项目，以表征生理活动控制的 通过分离法尼醇调节的基因来研究法尼醇和FXR。 解离 将小鼠肝细胞接种在培养皿中， 50 μ M法呢醇的0.5%甲醇溶液或单独与甲醇混合，持续3、8或17分钟 小时 从处理的细胞中分离RNA并用于编程cDNA 合成. 使用PCR扩增来自每个样品的cDNA。 聚合酶链反应和特异性寡核苷酸引物组。 三个不同的法尼醇调节基因片段被鉴定， 超过一百个引物组组合。 基因片段是 目前正在进行DNA测序 作为第二个方面， 项目，外显子对应的第二个一半的FXR DNA结合 从小鼠基因组DNA文库中分离结构域。 这种FXR编码 通过取代磷酸甘油酸激酶破坏外显子 启动子驱动的新霉素基因元件。 疱疹病毒胸苷激酶 将基因引入1.8内切酶对FXR基因组的下游， 片段以产生用于选择的靶向载体 FXR破坏的胚胎干细胞。 囊胚将注射 用这些细胞来制造具有FXR争议基因的动物。

FXR was previously identified as a nuclear receptor activated by molecules possessing farnesyl backbones. We have continued work on this project to characterize the physiological activities controlled by farnesoids and FXR by isolating farnesol-regulated genes. Dissociated mouse liver cells were seeded in culture dishes and incubated either with 50 uM farnesol in 0.5% methanol or with methanol alone for 3, 8, or 17 hours. RNAs were isolated from treated cells and used to program cDNA synthesis. The cDNAs from each sample were amplified using the polymerase chain reaction and specific oligonucleotide primer sets. Three distinct farnesol-regulated gene fragments were identified from more than one hundred primer set combinations. The gene fragments are presently being subjected to DNA sequencing. As a second aspect of this project, an exon corresponding to the second half of the FXR DNA-binding domain was isolated from a mouse genomic DNA library. This FXR-encoding exon was disrupted by substituting a phosphoglycerate kinase promoter-driven neomycin gene element. The herpes virus thymidine kinase gene was introduced downstream of a 1.8 kilobase pair FXR genomic fragment to create a targeting vector to be used for selecting FXR-disrupted embryonal stem cells. Blastocysts will be injected with these cells to create animals with FXR-disputed genes.