Elling Ulrich, Wimmer Reiner A, Leibbrandt Andreas, Burkard Thomas, Michlits Georg, Leopoldi Alexandra, Micheler Thomas, Abdeen Dana, Zhuk Sergei, Aspalter Irene M, Handl Cornelia, Liebergesell Julia, Hubmann Maria, Husa Anna-Maria, Kinzer Manuela, Schuller Nicole, Wetzel Ellen, van de Loo Nina, Martinez Jorge Arturo Zepeda, Estoppey David, Riedl Ralph, Yang Fengtang, Fu Beiyuan, Dechat Thomas, Ivics Zoltán, Agu Chukwuma A, Bell Oliver, Blaas Dieter, Gerhardt Holger, Hoepfner Dominic, Stark Alexander, Penninger Josef M
Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria.
Vienna Biocenter Core Facilities, Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria.
Nature. 2017 Oct 5;550(7674):114-118. doi: 10.1038/nature24027. Epub 2017 Sep 27.
The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.
直接揭示基因对特定表型的贡献的能力是生物学研究的基础。然而，表面上同质的细胞群体表现出很大的克隆变异，这可能会混淆分析并破坏可重复性。在这里，我们使用基因组饱和诱变创建了一个生物样本库，其中包含超过10万个个体单倍体小鼠胚胎干细胞系，针对16970个基因进行了基因条形码标记、条件性和可逆性突变。据我们所知，这个单倍体库是迄今为止最大的半合子/纯合子突变小鼠胚胎干细胞资源，可供所有研究人员使用。可逆诱变通过直接在姐妹细胞中对基因组进行功能注释来克服克隆变异。我们在反向遗传筛选中使用单倍体库来研究小鼠胚胎干细胞中必需基因的时间分辨率，并识别控制血管生成和血管谱系特化的新基因。此外，使用单倍体库进行的全基因组正向筛选确定磷脂酶A2G16是鼻病毒（导致普通感冒）细胞毒性所需的宿主因子。因此，来自单倍体库的克隆与可逆技术的结合能够实现基因组的高通量、可重复的功能注释。
