Balmer Jasmin, Ji Rui, Ray Thomas A, Selber Fabia, Gassmann Max, Peachey Neal S, Gregg Ronald G, Enzmann Volker
Department of Ophthalmology, Inselspital, University of Bern, Switzerland ; Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland.
Department of Biochemistry and Molecular Biology, University of Louisville, KY.
Mol Vis. 2013 Dec 31;19:2615-25. eCollection 2013.
To identify the mutation responsible for an abnormal electroretinogram (ERG) in a transgenic mouse line (tg21) overexpressing erythropoietin (Epo). The tg21 line was generated on a mixed (C3H; C57BL/6) background and lacked the b-wave component of the ERG. This no-b-wave (nob) ERG is seen in other mouse models with depolarizing bipolar cell (DBC) dysfunction and in patients with the complete form of congenital stationary night blindness (cCSNB). We determined the basis for the nob ERG phenotype and screened C3H mice for the mutation to evaluate whether this finding is important for the vision research community.
ERGs were used to examine retinal function. The retinal structure of the transgenic mice was investigated using histology and immunohistochemistry. Inverse PCR was performed to identify the insertion site of the Epo transgene in the mouse genome. Affected mice were backcrossed to follow the inheritance pattern of the nob ERG phenotype. Quantitative real-time PCR (qRT PCR), Sanger sequencing, and immunohistochemistry were used to identify the mutation causing the defect. Additional C3H sublines were screened for the detected mutation.
Retinal histology and blood vessel structure were not disturbed, and no loss of DBCs was observed in the tg21 nob mice. The mutation causing the nob ERG phenotype is inherited independently of the tg21 transgene. The qRT PCR experiments revealed that the nob ERG phenotype reflected a mutation in Gpr179, a gene involved in DBC signal transduction. PCR analysis confirmed the presence of the Gpr179(nob5) insertional mutation in intron 1 of Gpr179. Screening for mutations in other C3H-derived lines revealed that C3H.Pde6b(+) mice carry the Gpr179 (nob5) allele whereas C3H/HeH mice do not.
We identified the presence of the Gpr179(nob5) mutation causing DBC dysfunction in a C3H-derived transgenic mouse line. The nob phenotype is not related to the presence of the transgene. The Gpr179(nob5) allele can be added to the list of background alleles that impact retinal function in commonly used mouse lines. By providing primers to distinguish between Gpr179 mutant and wild-type alleles, this study allows investigators to monitor for the presence of the Gpr179(nob5) mutation in other mouse lines derived from C3H.
在过表达促红细胞生成素（Epo）的转基因小鼠品系（tg21）中鉴定导致异常视网膜电图（ERG）的突变。tg21品系是在混合（C3H；C57BL/6）背景下产生的，其ERG缺乏b波成分。这种无b波（nob）ERG在其他具有去极化双极细胞（DBC）功能障碍的小鼠模型以及先天性静止性夜盲（cCSNB）完全型患者中也可见。我们确定了nob ERG表型的基础，并在C3H小鼠中筛选该突变，以评估这一发现对视觉研究领域是否重要。
使用ERG检查视网膜功能。利用组织学和免疫组织化学研究转基因小鼠的视网膜结构。进行反向PCR以鉴定Epo转基因在小鼠基因组中的插入位点。将受影响的小鼠回交以追踪nob ERG表型的遗传模式。使用定量实时PCR（qRT PCR）、桑格测序和免疫组织化学来鉴定导致缺陷的突变。对其他C3H亚系进行检测突变的筛选。
视网膜组织学和血管结构未受干扰，在tg21 nob小鼠中未观察到DBC丢失。导致nob ERG表型的突变独立于tg21转基因遗传。qRT PCR实验表明，nob ERG表型反映了Gpr179基因的突变，该基因参与DBC信号转导。PCR分析证实了Gpr179第1内含子中存在Gpr179（nob5）插入突变。对其他源自C3H的品系进行突变筛选发现，C3H.Pde6b（+）小鼠携带Gpr179（nob5）等位基因，而C3H/HeH小鼠则不携带。
我们在源自C3H的转基因小鼠品系中鉴定出导致DBC功能障碍的Gpr179（nob5）突变的存在。nob表型与转基因的存在无关。Gpr179（nob5）等位基因可添加到影响常用小鼠品系视网膜功能的背景等位基因列表中。通过提供区分Gpr179突变体和野生型等位基因的引物，本研究使研究人员能够监测源自C3H的其他小鼠品系中Gpr179（nob5）突变的存在。
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