ANALYSIS OF INSERTIONAL MUTATIONS IN TRANSGENIC MICE

具有插入突变的转基因小鼠构成了潜在的丰富 分子标记的重要基因的来源。我们选择了两个 插入突变以进行更仔细的检查。带插入物的小鼠 11号染色体(MAP位置58)显示轻度发育异常 以椎骨融合和椎骨分裂为特征的椎骨 尤其是在胸部和腰部。这种表型是 这让人想起脊椎动物pax 1的突变所造成的 转录因子，其基因位于2号染色体上。它是 可以想象，11号染色体上中断的基因是 Pax 1难以捉摸的靶基因。我们已经鉴定了一段基因组DNA 在插入的转基因两侧。相应的序列被表达 在肌肉和心脏等成人组织中发现了一条2.8kb的mRNA。 目前，我们正在鉴定另一段侧翼DNA，可能 允许我们绘制任何可能伴随着 插入。然后，我们将着手确定该地区的文字记录 11号染色体上与表型有关的基因。 第二个插入发生在遗传定义的mI基因座上 小鼠6号染色体。MI突变的特征是三联体、丢失 有毛色素、小红眼和听力障碍。平凡的 这些缺陷的分母可能是不发达或丧失 遍布全身的功能性黑素细胞。事实上，对 这些转基因小鼠的内耳显示，所谓的纹状体 血管中没有黑色素细胞，这一现象与 外毛细胞退化。我们已经确认了两件 插入的转基因两侧的基因组DNA。这些序列是 存在于6.6 kb的单个基因组片段上，并定义了 另一种在mi基因发生突变的小鼠。其中一个探测器检测到 在Northern blotts上，在成人皮肤和 黑素细胞系，而在许多其他器官中不存在。因此，这个信使核糖核酸 可能代表了mi基因的转录本。 分析转基因插入突变的重要性不是 不仅在于对发育重要基因的分析，而且还在于 人类遗传性疾病分子特征的可能性 类似的现象学。脊椎异常是由 11号染色体的插入让人联想到某些人类脊椎 畸形，而mi突变是一种特殊形式的 人类瓦登堡综合征的特征是听力障碍和 色素异常。

Transgenic mice with insertional mutations constitute a potentially rich source of molecularly tagged important genes. We have selected two insertional mutations for closer scrutiny. Mice with an insertion on chromosome 11 (map position 58) display a mild developmental abnormality of the vertebrae characterized by vertebral fusions and split vertebrae particularly in the thoracic and lumbar regions. This phenotype is reminiscent of that created by mutations in pax 1, a vertebrate transcription factor, whose gene, however, is on chromosome 2. It is conceivable that the interrupted gene on chromosome 11 is one of the elusive target genes of Pax 1. We have identified a piece of genomic DNA flanking the inserted transgene. The corresponding sequence is expressed on a 2.8 kb mRNA found in adult tissues such as muscle and heart. Currently, we are identifying a second piece of flanking DNA that might allow us to map the extent of any deletion that may have accompanied the insertion. We will then proceed to identify the transcript of the region of chromosome 11 that is responsible for the phenotype. A second insertion occurred into the genetically defined mi locus on mouse chromosome 6. Mutations in mi are characterized by the triad, loss of coat pigment, small red eyes, and hearing deficiency. The common denominator of these defects may be the underdevelopment, or loss, of functional melanocytes throughout the body. In fact, analysis of the inner ear of these transgenic mice has revealed that the so-called stria vascularis is free of melanocytes, a phenomenon that is associated with degeneration of the outer hair cells. We have identified two pieces of genomic DNA flanking the inserted transgenes. These sequences are present on a single genomic fragment of 6.6 kb and define an RFLP in another line of mice with a mutation at mi. One of these probes detects on Northern blots approximately 7.0 kb mRNA found in adult skin and melanocyte cell lines, and not in many other organs. Thus, this mRNA likely represents a transcript of the mi gene. The importance of the analysis of transgenic insertional mutations not only lies in the analysis of developmentally important genes but also in the potential to molecularly characterize human genetic diseases with similar phenomenology. The vertebral abnormality defined by the chromosome 11 insertion is reminiscent of certain human vertebral malformations, and the mi mutation is a model for particular forms of human Waardenburg syndrome characterized by hearing deficiency and pigment abnormalities.