Ramalho José S, Anders Ross, Jaissle Gesine B, Seeliger Mathias W, Huxley Clare, Seabra Miguel C
Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London, SW7 2AZ, UK.
BMC Cell Biol. 2002 Oct 28;3:26. doi: 10.1186/1471-2121-3-26.
Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process.
To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous beta-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.
We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.
转基因小鼠已被证明是研究正常和病理基因功能的强大系统。在此，我们描述了一项尝试，旨在生成脉络膜视网膜病变（CHM）的转基因小鼠模型，CHM是一种由Rab护送蛋白-1（REP1）基因突变引起的迟发性X连锁视网膜变性疾病。REP1是Rab geranylgeranylation机制的一部分，这种修饰对于Rab在膜运输中的功能至关重要。CHM患者中REP1的缺失可能通过其对Rab蛋白的影响引发视网膜变性。我们之前报道过Rab27a是CHM淋巴母细胞中受影响最大的Rab，并推测Rab27a（可能还有其他一些Rab GTP酶）的选择性功能障碍在视网膜变性过程中起关键作用。
为了研究这一假设，我们生成了几系显性负性、组成型激活和野生型Rab27a（以及Rab27b）转基因小鼠，其表达由色素细胞特异性酪氨酸酶启动子或普遍存在 的β-肌动蛋白启动子驱动。在表达野生型或组成型激活Rab27a和Rab27b的转基因系中观察到高水平的mRNA和蛋白质。然而，仅表达了适度水平的转基因蛋白。脉冲追踪实验表明，显性负性蛋白而非组成型激活或野生型蛋白会迅速降解。一致地，在我们的转基因系中未观察到明显的表型。毛色正常，表明Rab27a活性正常。通过眼底镜检查、血管造影、视网膜电图和组织学确定的视网膜功能也正常。
我们认为，显性负性突变Rab27蛋白在体内的不稳定性使得无法使用这种方法来生成由Rab27 GTP酶引起的疾病的小鼠模型。
