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

为了鉴定遗传性疾病Bloom综合征（BS）患者细胞中携带突变基因的染色体，我们通过微细胞融合进行了染色体转移实验。来源于正常人成纤维细胞的含有单个psv2neo标记的15号染色体的小鼠A9细胞作为人染色体转移的供体细胞。纯化的A9微细胞与sv40转化细胞系GM8505和BS成纤维细胞自发转化细胞系GM1492E融合。BS细胞的特点是姐妹染色单体交换（SCE）频率极高。因此，我们研究了染色体转移后微细胞中SCEs频率的恢复。通过15号染色体的转移，两株细胞系GM8505、GM1492E的SCEs发生率均得到恢复。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。因此，需要进一步的RFLP分析来详细定位BS基因。染色体转移和SCE分析用colcolid处理含有单个人染色体15的小鼠A9细胞，诱导微核，离心去核。将微细胞与2株BS细胞系融合。孵育24小时后，将细胞置于含有G418的培养皿中。获得了G418抗性克隆。在5-溴-2-脱氧尿苷（BrdU）处理开始后24-27 h检测sce；在化学处理的同时加入BrdU （10ug/ml）。FPG染色并对SCEs进行评分。

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.