Generation, characterization, and molecular cloning of the Noerg-1 mutation of rhodopsin in the mouse.
Generation, characterization, and molecular cloning of the Noerg-1 mutation of rhodopsin in the mouse.
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小鼠视紫红质 Noerg-1 突变的生成、表征和分子克隆。
DOI：
10.1017/s0952523805225117
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发表时间：
2005
影响因子：
1.9
通讯作者：
Mullins,Robert
中科院分区：
文献类型：
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作者：
Pinto,LawrenceH;Vitaterna,MarthaH;Shimomura,Kazuhiro;Siepka,SandraM;McDearmon,ErinL;Fenner,Deborah;Lumayag,StephenL;Omura,Chiaki;Andrews,AnneW;Baker,Matthew;Invergo,BrandonM;Olvera,MarissaA;Heffron,Edward;Mullins,Robert
我们使用诱变剂N-乙基-N-亚硝基脲（ENU）对C57 BL/6 J小鼠进行全基因组诱变，并使用视网膜电图和眼底照相术筛选第三代（G3）后代的视觉系统改变。当在12-14周龄时测试时，一个谱系中的几只小鼠显示出视网膜变性的特征：没有可记录的视网膜电图（ERG）、视网膜血管衰减和眼底的斑点状色素沉着。组织学研究表明，视网膜经历光感受器变性，外核层细胞核的凋亡损失，但在明视条件下使用视动反应测量的视敏度持续存在，尽管有相当大的光感受器损失。Noerg-1突变在后代中表现为常染色体显性遗传模式。对出生后早期小鼠的研究表明，在部分功能性杆形成后发生变性。通过将C57 BL/6 J突变体与DBA/2 J或BALB/cJ小鼠杂交产生N2代，然后使用SSLP和SNP标记在多个位点确定N2后代的ERG表型和基因型，将Noerg-1突变遗传定位到染色体6。用一组紧密间隔的标记完成精细作图。从112.8 Mb到115.1 Mb的非重组区域被确定，包括视紫红质（Rho）编码区。在Rho基因中发现了从G到A的单核苷酸转换，预测其导致在椎间盘内环中位置110处Tyr替换为Cys。这种突变已在常染色体显性视网膜色素变性（RP）患者中发现，并导致体外表达的视紫红质的错误折叠。因此，ENU诱变能够复制人类患者中发生的突变，并且可用于产生人类遗传性眼病的从头模型。此外，小鼠基因组序列和广泛的DNA多态性的可用性使得该基因的快速鉴定成为可能，这表明使用ENU诱导的突变进行功能基因鉴定现在对于各个实验室是实用的。
We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12–14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A nonrecombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.