正向遗传学是筛选由化学诱变产生的显示特定表型的突变体，再确定突变基因的遗传分析方法。近年来外显子测序技术的快速发展使突变基因定位这一技术瓶颈得到克服，使得单个实验室进行筛选变得可行。.本项目计划运用正向遗传筛选寻找调节胚胎发育和干细胞产生和维持的新基因。我们将用化学诱变剂ENU，随机诱发基因组上单个碱基的突变，以模拟人类疾病中的遗传变异。通过筛选发育缺陷的突变体，寻找调控胚胎发育的新因子；通过筛选调控Oct4基因表达及Oct4阳性细胞增殖、分化和迁移的突变，寻找与干细胞功能相关的基因。孙欣教授在利用小鼠模型研究人类出生缺陷遗传病方面有成功经验，为后续ENU筛选奠定了基础。徐国良实验室在细胞多能性的表观遗传调控研究方面有多年积累，过去几年中两个实验室保持紧密合作，相关成果已发表文章，同时制备了后续筛选需要的转基因小鼠，并获得一些突变体。该项目将为进一步利用正向遗传研究生物学界重大问题奠定基础

A vast majority of current mammalian genetic studies utilize the reverse genetic approach, which relies on careful selection of candidate genes for functional interrogation. These studies are constrained by existing knowledge that provides the basis of candidate gene selection. In contrast, the forward genetic approach makes the simple hypothesis that if a gene is essential for a biological process, altering the function of this gene will reveal a phenotype in the process. As no prior knowledge of gene expression or function is required, the forward genetic approach offers a non-biased avenue to uncover novel drivers of key biological processes..The power of forward genetic screens has been unequivocally demonstrated in studies of lower, and non-mammalian organisms. However in mammalian genetic research up until now, this approach has not been widely adopted due to difficulties in the identification of causal mutations. We advocate here that recent advancements in genome sequencing technology has effectively removed this roadblock, and has made it feasible for individual laboratories to carry out forward genetic screens in mice..We propose here to conduct forward genetic screens to identify novel mutations that alter normal embryo development and stem cell behavior. We have chosen to use a chemical-based mutagen N-ethyl-N-nitrosourea (ENU) to induce small genomic changes that mimic the majority of genetic lesions underlying human diseases. To study normal development, we have established a comprehensive phenotype list for the screen that mimic a large spectrum of human birth defects. To study stem cell behavior, we will focus on the cardinal pluripotency factor, Oct4, and screen for mutations that alter Oct4-expression, and the proliferation, survival, differentiation, or migration of Oct4-expressing cells. The two topics of the screen have been our shared interests for many years. .We argue that our collaborative team is ideally positioned to be at the forefront of using chemical-based forward genetic screens in mice to identify novel genetic players. 1) Dr. Sun is a leader on using mouse as a model to understand human birth defects. 2) Dr. Xu is a leader in the study of genetic and epigenetic control of pluripotency. 3) Both investigators are familiar with the detailed operations of chemical-based forward genetic screens through communications with the world leaders in this field. 4) Most importantly, the two laboratories have had a history of productive collaborations. In addition to joint publications, we have together conducted a fruitful pilot screen, which will ensure the success of the proposed screen. In addition, we have generated transgenic mouse reagents that are critical for the proposed next screen. Our long-term goal here is not only to identify novel controls of development and pluripotency, but also to demonstrate the power and feasibility of the forward genetic technology to other mammalian genetic researchers in China..