Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.
Department of Oncology, Department of Medicine, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA; Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA.
Methods. 2014 Aug 1;68(3):529-35. doi: 10.1016/j.ymeth.2014.05.011. Epub 2014 Jun 2.
Recent breakthrough discoveries have shown that committed cell fates can be reprogrammed by genetic, chemical and environmental manipulations. The germline of the nematode Caenorhabditis elegans provides a tractable system for studying cell fate reprogramming within the context of a whole organism. To explore the possibility of using C. elegans in high-throughput screens (HTS), we developed a high-throughput workflow for testing compounds that modulate cell fate reprogramming. We utilized puf-8; lip-1 mutants that have enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling. Wild-type C. elegans hermaphrodites produce both sperm and oocytes, and are thus self-fertile. However, puf-8; lip-1 mutants produce only sperm and are sterile. Notably, compounds that pharmacologically down-regulate MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling are able to reprogram germ cell fate and restore fertility to these animals. puf-8; lip-1 mutants provide numerous challenges for HTS. First, they are sterile as homozygotes and must be maintained as heterozygotes using a balancer chromosome. Second, homozygous animals for experimentation must be physically separated from the rest of the population. Third, a high quality, high-content assay has not been developed to measure compound effects on germ cell fate reprogramming. Here we describe a semi-automated high-throughput workflow that enables effective sorting of homozygous puf-8; lip-1 mutants into 384-well plates using the COPAS™ BIOSORT. In addition, we have developed an image-based assay for rapidly measuring germ cell reprogramming by measuring the number of viable progeny in wells. The methods presented in this report enable the use of puf-8; lip-1 mutants in HTS campaigns for chemical modulators of germ cell reprogramming within the context of a whole organism.
最近的突破性发现表明，通过遗传、化学和环境操作，可以重编程已分化细胞的命运。秀丽隐杆线虫的生殖系为在整个生物体背景下研究细胞命运重编程提供了一个易于处理的系统。为了探索在高通量筛选 (HTS) 中使用秀丽隐杆线虫的可能性，我们开发了一种用于测试调节细胞命运重编程的化合物的高通量工作流程。我们利用了 puf-8; lip-1 突变体，它们增强了 MPK-1（ERK 同源物）/MAP 激酶 (MAPK) 信号。野生型秀丽隐杆线虫雌雄同体既能产生精子又能产生卵子，因此具有自育能力。然而，puf-8; lip-1 突变体只产生精子，且不育。值得注意的是，能在药理学上下调 MPK-1（ERK 同源物）/MAP 激酶 (MAPK) 信号的化合物能够重编程生殖细胞命运并使这些动物恢复生育能力。puf-8; lip-1 突变体为 HTS 带来了诸多挑战。首先，它们作为纯合子是不育的，必须使用平衡染色体保持为杂合子。其次，用于实验的纯合动物必须与其余种群物理隔离。第三，尚未开发出高质量、高内涵的测定法来测量化合物对生殖细胞命运重编程的影响。在这里，我们描述了一种半自动高通量工作流程，该流程使用 COPAS™ BIOSORT 将纯合的 puf-8; lip-1 突变体有效分选到 384 孔板中。此外，我们还开发了一种基于图像的测定法，通过测量孔中存活后代的数量来快速测量生殖细胞重编程。本报告中介绍的方法使 puf-8; lip-1 突变体能够用于 HTS 实验，以筛选化学调节剂对整个生物体背景下生殖细胞重编程的影响。
