ISOLATION OF A CANDIDATE CDNA FOR THE GENE CAUSING RETINAL DEGENERATION IN THE RD MOUSE
ISOLATION OF A CANDIDATE CDNA FOR THE GENE CAUSING RETINAL DEGENERATION IN THE RD MOUSE
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DOI：
10.1073/pnas.86.24.9722
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发表时间：
1989-12-01
影响因子：
11.1
通讯作者：
FARBER, DB
中科院分区：
文献类型：
--
作者：
BOWES, C;DANCIGER, M;FARBER, DB
rd 小鼠的遗传性视网膜变性导致一种细胞类型（光感受器）的完全丧失。我们利用这种视觉细胞损失的优势，设计了一种基于消减杂交和差异杂交分离光感受器特异性 cDNA 的策略。筛选所得光感受器特异性 cDNA 库中 rd 基因的候选 cDNA，并鉴定了映射到小鼠 5 号染色体（已分配 rd 基因的染色体）的推定 rd cDNA。在 Northern 印迹上，候选 rd cDNA 与来自 9 至 11 天发育正常视网膜的 3.3 kb RNA 物种杂交，并且更微弱地与来自年龄匹配的 rd 视网膜的 3.6 kb RNA 物种杂交。杂交的 mRNA 大小有 0.3 千碱基的差异，表明 rd 小鼠中与候选 rd cDNA 相对应的基因发生了结构改变。用限制性内切酶消化并用候选 rd cDNA 探测后，检测到 rd/rd 和 +/+ 小鼠基因组 DNA 之间的多态性进一步支持了这一点。出生后第 1 天，在正常和患病视网膜中检测到与候选 rd cDNA 杂交的 mRNA 表达，但 rd 视网膜中的信号强度相当低。据我们所知，这是在 rd 视网膜中报道的最早的分子缺陷，该缺陷是在光感受器变性的任何表型迹象之前观察到的。
The inherited retinal degeneration of the rd mouse results in the exclusive loss of one cell type, the photoreceptors. We took advantage of this visual-cell loss to devise a strategy for the isolation of photoreceptor-specific cDNAs based on the use of subtractive and differential hybridizations. The resulting pool of photoreceptor-specific cDNAs was screened for a candidate cDNA for the rd gene, and a putative rd cDNA that maps to mouse chromosome 5, the chromosome to which the rd gene has been assigned, was identified. On Northern blots the candidate rd cDNA hybridizes a 3.3-kilobase RNA species from 9- to 11-day-old developing normal retina and, much more faintly, a 3.6-kb RNA species from age-matched rd retina. The 0.3-kilobase difference in the size of the mRNAs hybridized suggests that a structural alteration in the gene corresponding to the candidate of rd cDNA has occurrred in the rd mouse. This was further supported by the detection of polymorphisms between rd/rd and +/+ mouse genomic DNA after digestion with restriction endonucleases and probing with the candidate rd cDNA. Expression of mRNAs hybridized by the candidate rd cDNA is detected in normal and diseased retinas at postnatal day 1 but the signal intensity is considerably lower in the rd retina. To our knowledge, this is the earliest molecular defect reported in the rd retina that is observed prior to any phenotypic signs of photoreceptor degeneration.