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Chromosomal mupping of a gene responsible for Bloom syndrome via microcell-mediated chromosome Transfer

通过微细胞介导的染色体转移对导致布卢姆综合征的基因进行染色体修饰

基本信息

批准号：
04454539
负责人：
OSHIMURA Mitsuo
金额：
$ 2.5万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

In order to identify the human chromosome which carries a mutated gene in cells from patients with the hereditary disorder, Bloom syndrome (BS), we performed chromosome transfer experimets via microcell fusion. Mouse A9 cells containing a single copy of pSV2neo-tagged chromosome 15 derived from normal human fibroblasts served as donor cells for transfer of human chromosome. Purified A9 microcells were fused with SV40-transformed cell line (GM8505) and spontaneously transformed cell line (GM1492E) derived from BS fibroblasts.Cell from BS is chracterized by an extremely high frequency of sister chromatid exchange (SCE). Thus, we examined the restoration of SCEs frequency in the microcells with the transfer of chromosome. The SCEs incidence in the both cell lines (GM8505, GM1492E) were restored by the transfer of chromosome 15. Chromosome analyzes revealed that these clones contained the intact chromosome 15, rearranged chromosome 15 or none. Thus, further analyzes with RFLP are needed for detailed mapping of the BS gene.Chromosome Transfer and SCE analyzesMouse A9 cells containing a single human chromosaome 15 were treated with colcemid to induce micronuclei and were enucleated by centrifugation. The microcells were fused to 2 BS cell lines. After incubation for 24 hr, cells were plated into dishes with medium containing G418. G418 resistant clones were isolated.SCEs were examined 24-27 h after the initiation of 5-bromo-2-deoxyuridine (BrdU) treatment ; BrdU (10ug/ml) was added at the same times as the chemical treatment. Slides were stained by FPG treatment and SCEs were scored.

为了从遗传性疾病Bloom综合征（BS）患者的细胞中鉴定携带突变基因的人类染色体，我们通过微细胞融合进行了染色体转移实验。含有来源于正常人成纤维细胞的pSV 2neo标记的15号染色体的单拷贝的小鼠A9细胞用作转移人染色体的供体细胞。将纯化的A9细胞与SV 40转化的BS成纤维细胞系（GM 8505）和自发转化的BS成纤维细胞系（GM 1492 E）融合，BS细胞具有极高的姐妹染色单体交换（SCE）频率。因此，我们研究了随着染色体的转移，微细胞中SCE频率的恢复。两种细胞系（GM 8505，GM 1492 E）中SCE的发生率通过15号染色体的转移而恢复。染色体分析表明，这些克隆含有完整的15号染色体，重排的15号染色体或没有。染色体转移和SCE分析用秋水仙胺处理含有单个人染色体15的小鼠A9细胞以诱导微核，并通过离心去核。将微细胞与2个BS细胞系融合。孵育24小时后，将细胞接种到含有G418的培养基的培养皿中。用5-溴-2-脱氧尿苷（BrdU，10 μ g/ml）处理细胞24-27 h后，检测SCE。通过FPG处理对载玻片进行染色，并对SCE进行评分。