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在TK6和L5178Y细胞的背景突变频率:对错误纠正测序的影响
Jaime A Miranda1, Azra Dad1, Xuewei Qu1
1Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, USA.
Environmental and molecular mutagenesis
|July 15, 2025
概括
细胞培养中的背景突变可以扭曲用于突变性测试的纠错测序 (ECS) 的结果. 使用新鲜的细胞克隆可以最大限度地减少这个问题,提高突变性评估的准确性.
科学领域:
- 基因组学和分子生物学
- 毒理学和变异发生过程
背景情况:
- 错误纠正测序 (ECS) 方法对于检测超低频突变和评估突变性至关重要.
- 在不朽化的细胞培养物中积累的自发突变可以提高背景突变频率 (MF),可能会混ECS结果.
- 商用TK6和L5178Y细胞种群的背景MF比新得出的克隆种群高.
研究的目的:
- 调查在商用细胞系中背景突变对基于ECS的突变性评估的影响.
- 为了在ECS实验中比较商业上可用的细胞种群与新获得的克隆种群的性能.
- 评估不同数据分析方法对ECS结果在背景突变存在时的解释的影响.
主要方法:
- 基因组比较单细胞衍生克隆与父母TK6和L5178Y细胞培养物,以确定独特的突变.
- 培养商业上可用的和新鲜衍生的TK6细胞种群,其度在体外变异原体N4-氧丁的不同度.
- 使用HiFi测序,ECS方法分析突变性,并应用相对和绝对突变频率变化分析.
主要成果:
- 商用TK6和L5178Y细胞种群的背景MF (9 × 10−7和6 × 10−7突变/bp) 比新衍生的克隆 (0.5 × 10−7和1 × 10−7突变/bp) 高得多.
- 与商业种群相比,新获得的克隆TK6种群在暴露于变异原体时表现出较低的背景MF和较大的相对折叠MF增加.
- 绝对MF变化分析方法在商业和克隆细胞种群之间提供了比相对折叠增加分析更可比的结果.
结论:
- 在商用细胞系中背景MF的升高可能会影响体外ECS突变性实验的解释.
- 由于背景MFs较低,新衍生的克隆细胞种群为基于ECS的突变性评估提供了更可靠的基线.
- 数据分析方法的选择 (相对和绝对的MF变化) 可以影响不同细胞种群类型的结果的可比性.
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