Carette Jan E, Guimaraes Carla P, Varadarajan Malini, Park Annie S, Wuethrich Irene, Godarova Alzbeta, Kotecki Maciej, Cochran Brent H, Spooner Eric, Ploegh Hidde L, Brummelkamp Thijn R
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Science. 2009 Nov 27;326(5957):1231-5. doi: 10.1126/science.1178955.
Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.
在模式生物中进行的功能丧失型遗传筛选已经阐明了许多生物学过程，但是哺乳动物细胞的二倍体基因组阻碍了大规模的基因破坏。我们利用插入诱变开发了一种筛选方法，以在除8号染色体外所有染色体均为单倍体的人类细胞系中产生无效等位基因。通过这种方法，我们鉴定出了流感感染所必需的宿主因子以及编码白喉酰胺生物合成途径重要元件的基因，白喉酰胺是白喉毒素和外毒素A细胞毒性作用所必需的。我们还鉴定出了细胞致死性扩张毒素作用所需的基因，包括一种与该毒素相互作用的细胞表面蛋白。这种方法在概念和实践上都与单倍体酵母中的遗传方法相似。
