Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes

【原创】

1MitoCheck Project Group

2Gene Expression and

3Cell Biology/Biophysics Units, Structural and

4Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

5Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK.

6MaxPlanck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.

7Institute of Biochemistry, Swiss Federal Institute of Technology Zurich (ETHZ), Schafmattstrasse 18, CH-8093 Zurich, Switzerland.

8Leica Microsystems CMS GmbH, Am Friedensplatz 3, D-68165 Mannheim, Germany.

9European Bioinformatics Institute, European Molecular Biology Laboratory, Cambridge CB10 1SD, UK.

10Division of Theoretical Bioinformatics, German Cancer Research Center, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany.

11Institute for Molecular Pathology, Dr Bohr Gasse 7, A-1030 Vienna, Austria.

以上的研究机构合作用RNAi敲除HeLa cells的21000个蛋白，用于研究这些敲除的蛋白对有丝分裂的影响。相当于建了个瞬时表达的突变体库。好不容易获得了大量突变体，只有图像能够保存。不知有谁在做稳定遗传（转基因）的动物或人细胞突变体。

1.A (siRNA) library designed to uniquely target each gene with 2–3 independent sequences; The siRNAs in this library were tested individually and reduced the messenger RNAs of targeted genes to below 30% of original levels (to an average of 13%) for 97% of more than 1,000 genes tested.

2.To allow high-throughput phenotyping of each individual siRNA in triplicates by live-cell imaging, we used a previously established workflow for solid-phase transfection using siRNA microarrays coupled to automatic time-lapse microscopy. As a high-content phenotypic assay we chose to monitor fluorescent chromosomes in a human cell line stably expressing core histone 2B tagged with green fluorescent protein (GFP). After seeding on the siRNA microarrays, on average 67 (630) cells for each siRNA of the library were imaged in triplicates for 2 days, thus documenting many of their basic functions such as cell division, proliferation, survival and migration.