Daniel Philip B, Fernando Chathurini, Wu C-S Jenny, Marnane Rebecca, Broadhurst Ric, Mountjoy Kathleen G
Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1, New Zealand.
Mol Cell Endocrinol. 2005 Jul 15;239(1-2):63-71. doi: 10.1016/j.mce.2005.03.013.
The melanocortin 4 receptor (MC4R) plays a critical role in the regulation of energy homeostasis, and the MC4R knockout mouse and humans with MC4R defective mutations in only one allele indicate that there is a gene dosage effect. Alterations in gene expression levels for MC4R could, therefore, have significant effects on energy homeostasis. To begin to develop a mouse model for studies on MC4R promoter in situ we used approximately 1 kb mouse MC4R promoter together with 426 bp MC4R 5' UTR, previously shown to support basal expression of reporter gene transcription in cell lines with endogenous MC4R mRNA, and fused this DNA to a nuclear localized LacZ reporter gene. The construct was injected into pronuclei from FVB mice. Five transgenic lines were identified as carrying autosomal transgene insertions; three of these had significant beta-galactosidase staining in brain and in a few cells in the heart but not in kidney, liver, lung, gonadal fat or testis. The pattern of transgene expression in the brain differed markedly for the three lines, and in one of these lines was remarkably similar to endogenous MC4R mRNA expression observed using in situ hybridisation. In conclusion, approximately 1 kb mouse MC4R promoter is sufficient to direct gene expression to the brain including regions that express endogenous MC4R mRNA.
黑皮质素4受体（MC4R）在能量平衡调节中起关键作用，MC4R基因敲除小鼠以及仅一个等位基因存在MC4R缺陷突变的人类表明存在基因剂量效应。因此，MC4R基因表达水平的改变可能对能量平衡产生重大影响。为了开始建立一个用于原位研究MC4R启动子的小鼠模型，我们使用了大约1 kb的小鼠MC4R启动子以及426 bp的MC4R 5'非翻译区（UTR），之前已证明其能在内源性MC4R mRNA的细胞系中支持报告基因转录的基础表达，并将该DNA与核定位的LacZ报告基因融合。构建体被注射到FVB小鼠的原核中。鉴定出五个携带常染色体转基因插入的转基因系；其中三个在脑和心脏的少数细胞中有明显的β-半乳糖苷酶染色，但在肾脏、肝脏、肺、性腺脂肪或睾丸中没有。这三个系在脑中的转基因表达模式明显不同，其中一个系与使用原位杂交观察到的内源性MC4R mRNA表达非常相似。总之，大约1 kb的小鼠MC4R启动子足以将基因表达导向大脑，包括表达内源性MC4R mRNA的区域。
