微细胞介导人14号染色体自A9细胞转移至DT40细胞

Microcell-mediated Transfer of Human Chromosome 14 from A9 to DT40 Cell

为了获得含人14号染色体的DT40细胞,用于人抗体基因的表达研究.本研究利用微细胞介导的染色体转移技术,将A9细胞中的人14号染色体转移至DT40细胞中.首先,摸索秋水仙胺诱导A9细胞微核形成最佳浓度与最佳时间,以终浓度为10 mg/mL的细胞松驰素B破坏细胞骨架,离心分离微细胞,获得的微细胞依次经8μm、5μm、3μm滤膜过滤后与受体细胞DT40融合,细胞铺板后加入G418筛选.然后,对长出的抗性克隆进行基因组DNA检测及FISH杂交,分析人14号染色体在DT40杂合细胞克隆中的存在情况.结果显示,成功获得含人14号染色体的DT40(#14)细胞,三轮试验共获得抗性克隆30个,人14号染色体有效转移率为1×10-6.实验结果表明,人14号染色体完整的自A9细胞转移至DT40细胞,获得的DT40(#14)细胞可用于制备含人抗体基因的人类人工染色体,用于人抗体基因的表达研究.

With the technique of microcell-mediated chromosome transfer（MMCT）,we transferred the human chromosome 14 from A9 cells to DT40 cells.To optimize the colcemid concentration and exposure time for micronuclei formation,A9 cells were treated with cytochalasin B to disrupt microfilaments and stalled at the interphase prior to centrifugation.The microcells were collected and filtered sequentially through 8 μm,5 μm and 3 μm filters and fused with DT40 cell.The medium of 1.5mg /mL G418 was applied to select hybrid colonies.A total of 30 clones were PCR-tested to be positive and the clone DT40（#14） was further confirmed by FISH.The chromosome transfer efficiency was 1 × 10^-6 suggesting that human chromosome 14 cell could be transferred from A9 cells to DT40 cells efficiently.The obtained DT40（# 14） cells can be used for the experiments in human artificial chromosome construction and antibody expression.