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

为了在遗传性疾病Bloom综合征(BS)患者的细胞中识别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。小鼠A9细胞含有单个拷贝的pSV2neo标记的15号染色体，取自正常人成纤维细胞，作为人类染色体转移的供体细胞。将纯化的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)进行融合，发现BS细胞的姐妹染色单体互换(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，两个细胞系(GM8505、GM1492E)中SCES的发生率均得到恢复。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号染色体或不含15号染色体。染色体转移和姐妹染色单体交换分析含有单个人类15号染色体的小鼠A9细胞用秋水仙素诱导微核，并用离心法去核。将微细胞融合成2个BS细胞系。培养24小时后，将细胞接种于含G418的培养皿中。分离G418抗性克隆，在5-溴-2-脱氧尿苷(BrdU)处理24-27h后检测SCE，同时加入BrdU(10ug/ml)。对玻片进行FPG染色，并进行SCES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.