In vivo expansion of trinucleotide repeats yields plasmid and YAC constructs for targeting and transgenesis.
In vivo expansion of trinucleotide repeats yields plasmid and YAC constructs for targeting and transgenesis.
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三核苷酸重复的体内扩增产生用于靶向和转基因的质粒和YAC构建体。
DOI：
10.1016/s0378-1119(00)00508-4
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发表时间：
2000
期刊：
影响因子：
3.5
通讯作者：
LaSpada,AR
中科院分区：
文献类型：
--
作者：
Sopher,BL;Myrick,SB;Hong,JY;Smith,AC;LaSpada,AR
产生遗传性神经退行性疾病的小鼠模型是了解神经毒性机制和测试潜在疗法的重要一步。我们有兴趣创建 X 连锁脊髓和延髓肌萎缩症 (SBMA) 小鼠模型，SBMA 是一种由雄激素受体 (AR) 基因内 CAG 重复序列扩张引起的神经肌肉疾病。为了培育出在其一生中表现出 SBMA 表型的小鼠，我们决定获得携带 AR 基因的酵母人工染色体 (YAC)，并引入数量为 100 个或更多三联体的 CAG 重复突变。从未观察到具有超过 70 个 CAG 的 SBMA 患者；因此，我们选择在大肠杆菌体内扩增59个CAG重复序列。尽管我们开始使用涉及两个质粒共繁殖的重组范例来扩展这个重复序列，但我们没有观察到大规模的扩展。相反，我们能够利用复制不稳定性，在酵母整合质粒载体中逐步生成 100 至 200 个 CAG 的重复序列。在产生这些 CAG 重复序列的实验过程中，我们评估了重复方向、向量共同传播和 recA 函数在扩展过程中的作用。然后，我们使用其中一种酵母整合载体成功产生了携带 100 个 CAG 重复序列的 AR YAC 构建体。 AR YAC CAG100 将作为生产 SBMA 小鼠的宝贵试剂。
Production of mouse models of inherited neurodegenerative diseases is an important step towards understanding the mechanism of neurotoxicity and for testing potential therapies. We are interested in creating a mouse model for X-linked spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by expansion of a CAG repeat within the androgen receptor (AR) gene. To permit generation of mice that will show a SBMA phenotype within their life span, we decided to obtain a yeast artificial chromosome (YAC) carrying the AR gene and introduce CAG repeat mutations numbering 100 or more triplets. SBMA patients with more than 70 CAGs have never been observed; therefore, we chose to expand a 59 CAG repeat tract in vivo in Escherichia coli. Although we set out to expand this repeat tract using a recombination paradigm involving two plasmid co-propagation, we did not observe large expansions. We were instead able to incrementally generate repeat tracts from 100 to 200 CAGs in a yeast integrating plasmid vector by taking advantage of replication instability. In the course of our experiments that yielded these CAG repeat tracts, we evaluated the role of repeat orientation, vector co-propagation, and recA function on the expansion process. We then used one of the yeast integrating vectors to successfully produce an AR YAC construct carrying 100 CAG repeats. AR YAC CAG100 will serve as a valuable reagent for the production of a SBMA mouse.