ANALYSIS OF INSERTIONAL MUTATIONS IN TRANSGENIC MICE

转基因小鼠插入突变分析

• 批准号: 2579580
• 负责人: H ARNHEITER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2579580
• 关键词: Waardenburg syndrome; alleles; congenital ear disorder; congenital eye disorder; developmental genetics; gene expression; gene induction /repression; gene mutation; genetically modified animals; hearing disorders; laboratory mouse; melanocyte; microphthalmos; retinal pigment epithelium; transcription factor; transposon /insertion element

Many congenital abnormalities of sensory organs such as eye and ear malformations are the result of derailed development due to malfunctioning genes. We have recently discovered a basic-helix-loop- heli-zipper transcription factor gene, called microphthalmia (mi), whose mutations in mice are associated with smaller-than-normal eyes, inner ear deafness, and loss of coat pigmentation. We have also isolated the human homolog of this mouse gene, and it turns out that a particular form of human syndromic hearing impairment called Waardenburg syndrome IIa is in fact due to mutations in this gene. In mice, the common denominator of the abnormalities is the aberrant development or plain absence of pigment cells in eye, inner ear and skin, and the same may be true in humans. We have now determined the developmental expression profile of mi in wild type and mutant mouse embryos as well as their cultured melanocytes. In wild type mouse embryos, expression starts in cells in the outer layer of the developing optic cup and soon thereafter in a very small number of cells derived from the neural crest. A few hours after expressing mi, the majority of these cells start to express Trp2, a melanoblast marker, and thus they seem to be committed to the melanocyte lineage. Since in some locations mi expression is found in cells located still in the dorsal wall of the neural tube, commitment may start prior to emigration. In embryos homozygous for certain mutant alleles, neural crest-derived mi-expressing cells are hardly detectable, and staining for other melanoblast markers such as Trp2 remains negative. However, the retinal pigment layer cells stay in place and continue to express Trp2 but not Trp1 and tyrosinase, two other melanocyte markers that based on in vitro analyses are direct target genes of mi. Since after birth, mi is no longer expressed in pigment cells except in those of the hair bulbs, it appears that all abnormalities in eyes and ears may be determined prior to birth, whereas abnormalities in skin pigmentation may continue to develop during life. In a collaborative effort, we have also started to determine the precise molecular defects associated with different mutant alleles in mice and men to analyze the biochemical and biological consequences of these defects in vitro and in tissue culture cells. Interestingly, coexpression of different mutant forms of the Mi protein in compound heterozygotes may lead to milder or more severe abnormalities when compared to the corresponding homozygous mice. Future studies will show whether this observation can be explained solely on the basis of formation of dimers between different mutant forms of the Mi protein or whether it implies involvement of other, related basic helix- loop-helix-zipper transcription factors.

许多先天性感觉器官异常，如眼和耳 畸形是由于脱轨的发展， 基因故障 我们最近发现了一种基本的螺旋环 螺旋拉链转录因子基因，称为小眼症（mi），其 小鼠的突变与眼睛比正常小、内耳有关 耳聋和被毛色素丧失。 我们还分离出了 这种小鼠基因的同源物，事实证明，一种特殊形式的 人类综合征性听力损伤称为Waardenburg综合征IIa， 这是由于该基因的突变。在小鼠中， 这些异常是指 眼睛、内耳和皮肤中的色素细胞， 人类我们现在已经确定了 野生型和突变型小鼠胚胎以及它们的培养物中的mi 黑素细胞 在野生型小鼠胚胎中， 外层的发展光学杯，并很快在一个非常 少量细胞来源于神经嵴。 几小时后 当表达mi时，这些细胞中的大多数开始表达Trp 2， 黑素细胞标志物，因此它们似乎致力于黑素细胞 脉由于在某些位置，mi表达存在于位于 仍然在神经管的背壁，承诺可能会在之前开始 移民。 在某些突变等位基因纯合子的胚胎中，神经 嵴源性表达mi的细胞几乎检测不到， 其它成黑素细胞标志物如Trp 2保持阴性。 但 视网膜色素层细胞保持原位并继续表达Trp 2 但Trp 1和酪氨酸酶却没有，这两种黑素细胞标志物是基于 体外分析是MI的直接靶基因。 出生后，我 不再在色素细胞中表达， 灯泡，似乎所有异常的眼睛和耳朵可能是 在出生前确定，而皮肤色素沉着异常可能 在生活中不断发展。在合作努力中，我们还 开始确定与 不同的突变等位基因在小鼠和人，以分析生化和 这些缺陷在体外和组织培养中的生物学后果 细胞 有趣的是，共表达不同突变形式的Mi 复合杂合子中的蛋白质可能导致轻度或重度 与相应的纯合子小鼠相比， 未来 研究将表明，这一观察结果是否可以单独解释， Mi的不同突变形式之间二聚体形成的基础 蛋白质或它是否意味着涉及其他相关的基本螺旋- 环螺旋拉链转录因子。