ANALYSIS OF INSERTIONAL MUTATIONS IN TRANSGENIC MICE

转基因小鼠插入突变分析

• 批准号: 3860872
• 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/3860872
• 关键词: bacteriophage lambda; congenital skeletal disorder; developmental genetics; evoked potentials; gene expression; gene mutation; genetic library; genetic mapping; genetically modified animals; hearing disorders; laboratory mouse; microphthalmos; molecular cloning; nucleic acid probes; nucleic acid sequence; pigmentation disorders; spina bifida; syndrome; transfection; virus genetics; virus infection mechanism

Insertion of transgenes into the mouse genome occasionally results in disruption of important endogenous genes. When bred to transgene homozygosity, such transgenic mice may exhibit mutant phenotypes resulting from improper functioning of the endogenous gene. We have encountered two different insertional mutants exhibiting developmental abnormalities. One line of mice (line 2627) harbors approximately 15 copies of a transgene on chromosome 11. The phenotype of this line is characterized by hemivertebrae, vertebrogenic spina bifida occculta, fusions of vertebral bodies, and short, kinky tails. This phenotype is reminiscent of undulated, a mutation in the paired-box of the Paxl protein encoded by the paxl gene on chromosome 2. Since Paxl and the gene with the transgene insertion map to different chromosomes, yet produce similar phenotypes when mutated, it is possible that the two gene products act along the same metabolic pathway. To clone the endogenous gene on chromosome 11, we have prepared genomic libraries in lambda phage and are currently screening them for the presence of DNA sequences flanking the transgene. A second line of transgenic mice (VGA 9) harbors approximately 50 copies of a transgene on chromosome 6. Homozygous transgenics show a complete loss of skin, complete or near-complete loss of eve pigmentation, microphthalmia, and a severe hearing deficiency of at least 50 dB as analyzed by brain stem auditory evoked potential measurements done in collaboration with Dr. Kenneth Grundfast from the LMO, NIDCD. The hearing deficiency may be the consequence of an underdevelopment of the stria vascularis of the cochlea where the intermediate cell layer with its melanin-producing cells is missing. Thus, the insertion affects, among other cell lineages, the proper development of the neural crest-- derived pigment cells. It is allelic with, and phenotypically similar to, the mutation mi (not cloned), and shares features with two other well characterized mutations, W (a mutation in the receptor kinase c-kit), and S/ (a mutation in the growth factor steel). The overlap of phenotypes suggests that these three mutations and the VGA 9 insertion affect genes of the same metabolic pathway, Of particular interest is the connection between VGA 9 and the human Waardenburg syndrome, a syndrome characterized by hereditary deafness and pigment abnormalities. Identifying the gene affected in the transgenics may provide molecular probes useful to characterize patients with this syndrome, and may help to understand the pathogenesis of this disease.

将转基因插入小鼠基因组中偶尔会导致 破坏重要的内源基因。当被培育成转基因时 纯合性，这样的转基因小鼠可能表现出突变的表型 由内源性基因的不正常功能引起的。我们有 遇到两个不同的插入突变体，表现出发育 异常现象。一条老鼠线(2627线)藏匿大约15只 11号染色体上的一个转基因拷贝。这个品系的表型是 以半椎体、椎源性隐性脊柱裂为特征， 融合的椎体和短而扭曲的尾巴。这种表型是 让人想起波状，Paxl配对盒中的一个突变 由2号染色体上的pax1基因编码的蛋白质。由于pax1和基因 将转基因插入图谱映射到不同的染色体，但产生 当表型相似时，两个基因有可能发生突变 产品沿着相同的代谢途径起作用。克隆内源性的 11号染色体上的基因，我们已经在Lambda噬菌体中建立了基因组文库 目前正在对它们进行DNA序列的筛选 在转基因的侧翼。 第二种转基因小鼠(VGA-9)含有大约50个拷贝。 6号染色体上的转基因。纯合子转基因显示 皮肤脱落，完全或几乎完全丧失夏娃色素沉着， 小眼炎，以及至少50分贝的严重听力缺陷 脑干听觉诱发电位测量结果分析 与NIDCD LMO的Kenneth Grundfast博士合作。这个 听力障碍可能是大脑发育不良的结果。 耳蜗纹的血管纹，中间细胞层 它的黑色素产生细胞不见了。因此，插入影响， 在其他细胞谱系中，神经脊的适当发育-- 衍生色素细胞。它等位基因与，表型相似 To，突变mi(未克隆)，并与另外两个井共享特征 特征性突变，W(受体激酶c-kit的突变)，以及 S/(生长因子钢的突变)。表型的重叠 提示这三个突变和VGA 9插入会影响基因 在相同的代谢途径中，特别有趣的是 在VGA 9和人类Waardenburg综合征之间 以遗传性耳聋和色素异常为特征的。 识别转基因中受影响的基因可能会提供分子 有助于确定患有这种综合征的患者的特征，并可能有助于 了解该病的发病机制。