微细胞介导人14号染色体自A9细胞转移至DT40细胞

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.

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

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.