本重点室黄粤课题组研究论文“Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens” 于2017年9月28日在线发表于《Nucleic Acids Research》（doi: 10.1093/nar/gkx857）。 

        利用哺乳动物细胞进行的遗传筛选工作已经被广泛的用于揭示基因的生物学功能，但哺乳动物细胞基因组的二倍体性阻碍了基因纯合突变体的获得，最近单倍体胚胎干细胞（Embryonic Stem Cell, ESC）的成功分离很好地解决了这一问题。在本研究中，我们利用小鼠单倍体ESC和PiggyBac（PB）转座子构建了矩阵式突变体文库，其中85% 以上的克隆是基因纯合突变体。我们首先利用该文库进行了正向选择性筛选，通过对2024个突变体克隆进行了分化相关基因的筛选，获得了40个与分化缺陷相关的基因，利用回复实验和CRISPR-Cas9技术在二倍体ESC中突变相关基因确定了相关基因和分化缺陷表型间的因果关系。由于负性选择性筛选是鉴定丢失的克隆，虽然混合文库筛选结合二代测序技术通过量化筛选前后所有的突变可以鉴定部分丢失的克隆，但混合文库中不同突变细胞之间的相互影响会干扰最后筛选的数据，而矩阵式突变体文库很好的避免了这个问题。在本研究中，我们进行了诱导DNA双链断裂药物-阿霉素(Doxorubicin)敏感性增加的遗传筛选，利用高内涵筛选仪从1152个突变克隆中获得了13个药物敏感性增加的克隆，其中定位到编码基因中有6个，回复克隆对阿霉素敏感性得到回复确定了相关基因与与药物敏感性之间的关系。因此，我们建立的矩阵式基因突变文库扩展了遗传筛选的应用，具有广泛的应用前景，并且建立相关网站可以为同行提供文库服务。

         有趣的是，国际著名学术期刊《Nature》于10月5日也发表了一篇研究内容相似的研究论文“A reversible haploid mouse embryonic stem cell biobank resource for functional genomics”，来自奥地利Josef Penninger教授研究组。 黄粤课题组的刘光助理研究员、王雪博士（2017年毕业）和刘语方博士（2015年毕业）为该研究论文的共同第一作者，课题组的张美丽助理研究员、贾玉艳助理研究员和北京生命科学研究所的蔡涛研究员、沈志荣研究员也参与该研究；黄粤研究员为本文通讯作者。该研究工作得到了科技部重点研发计划（2016YFA0100103）、中国医学科学院医学与健康科技创新工程（2016-I2M-3-002）及国家自然科学基金（2013CB967002和31671410）等项目的资助。 文章网址：https://academic.oup.com/nar/article/4259023 

[摘要]：Forward genetic screens using mammalian embryonic stem (ES) cells have identified genes required for numerous cellular processes. However, loss-off-function screens are more difficult to conduct in diploid cells because, in most cases, both alleles of a gene must be mutated to exhibit a phenotype. Recently, mammalian haploid ES cell lines were successfully established and applied to several recessive genetic screens. However, all these screens were performed in mixed pools of mutant cells and were mainly based on positive selection. In general, negative screening is not easy to apply to these mixed pools, although quantitative deep sequencing of mutagen insertions can help to identify some ‘missing’ mutants. Moreover, the interplay between different mutant cells in the mixed pools would interfere with the readout of the screens. Here, we developed a method for rapidly generating arrayed haploid mutant libraries in which the proportion of homozygous mutant clones can reach 85%. After screening thousands of individual mutant clones, we identified a number of novel factors required for the onset of differentiation in ES cells. A negative screen was also conducted to discover mutations conferring cells with increased sensitivity to DNA double strand breaks induced by the drug doxorubicin. Both of these screens illustrate the value of this system.

Figure 1. Generation, characterization and applications of the mutant haploid ES cell library. 
(A) Structure of the vector PBDGTV for gene trap. The vector is shown integrated into an intron, with purple boxes representing exons of the disrupted gene. PBDGTV can trap genes transcribed in either direction. SA, splice acceptor; PB5 and PB3, PB repeats; pA: SV40 ployA; Dom3z and Ccdc107: mutagenic units with the terminal and penultimate exons of each gene. 
(B) The experimental scheme to generate the arrayed mutant haploid ES cell library. 
(C) Southern blot analysis of gene-trap clones reveals the host/transposon junction fragment in each clone. SI: Stu I; probe, a 550-bp PCR fragment from PB3 and Ccdc107 in PBDGTV. Black triangles indicate the endogenous
Ccdc107. The lower table is the summary of copy number per clone in the arrayed library. 
(D) The distribution rates of PB (piggyBac) transposon in the mutant library. The transposon landed in 45% intergenic and 55% intragenic regions, with a high frequency of integration into introns. 
(E) Splinkerette PCR combined with massively parallel sequencing to identify the genome coverage and distribution rates of PB transposon in the mutant library. 
(F) Homozygosity analysis of individual mutant ES cell clones. S, mutant sample; C, wild-type haploid ES cells, AGH-OG-3. Heterozygous mutants are marked by red color.
(G) In the arrayed libraries, cells are arranged in multi-well plates with a single mutant gene per well. Arrayed screens can be conducted using automation for the handling of many plates and a much wider range of cellular phenotypes can be investigated using fluorescence, luminescence and high-content image analysis, as each well has a single genetic perturbation. In this study, we use the arrayed homozygous mutant libraries to conduct a positive genetic screen for identifying ‘exit-from-pluripotency’ factors and a negative genetic screen for mutations conferring increased sensitivity to the DNA double strand break (DSB)-inducing drug Doxorubicin.