Pinto Lawrence H, Vitaterna Martha H, Shimomura Kazuhiro, Siepka Sandra M, McDearmon Erin L, Fenner Deborah, Lumayag Stephen L, Omura Chiaki, Andrews Anne W, Baker Matthew, Invergo Brandon M, Olvera Marissa A, Heffron Edward, Mullins Robert F, Sheffield Val C, Stone Edwin M, Takahashi Joseph S
Department of Neurobiology and Physiology and Center for Functional Genomics, Northwestern University, Evanston, Il 60208, USA.
Vis Neurosci. 2005 Sep-Oct;22(5):619-29. doi: 10.1017/S0952523805225117.
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 non-recombinant 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.
我们使用诱变剂N-乙基-N-亚硝基脲（ENU）对C57BL/6J小鼠进行全基因组诱变，并使用视网膜电图和眼底照相术对第三代（G3）后代的视觉系统改变进行筛选。在一个谱系中的几只小鼠在12 - 14周龄时进行测试时表现出视网膜变性的特征：无记录到的视网膜电图（ERG）、视网膜血管变细以及眼底出现斑点状色素沉着。组织学研究表明，视网膜经历光感受器变性，外核层细胞核凋亡性丢失，但尽管光感受器大量丧失，在明视觉条件下使用视动反应测量的视力仍然存在。Noerg-1突变在后代中表现出常染色体显性遗传模式。对出生后早期小鼠的研究表明，变性发生在部分功能性视杆细胞形成之后。通过将C57BL/6J突变体与DBA/2J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突变基因定位到6号染色体上。使用一组紧密间隔的标记完成了精细定位。确定了一个从112.8 Mb到115. Mb的非重组区域，该区域包含视紫红质（Rho）编码区。在Rho基因中发现了一个从G到A的单核苷酸转换，预计这会导致在盘内环的第110位由半胱氨酸替代酪氨酸。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，并且对于生成人类遗传性眼病的全新模型很有用。此外，小鼠基因组序列的可用性和广泛的DNA多态性使得快速鉴定该基因成为可能，这表明使用ENU诱导的突变进行功能基因鉴定现在对各个实验室来说是可行的。
