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Enabling mammalian in vivo forward genetic screens based on cell morphology

实现基于细胞形态的哺乳动物体内正向遗传筛选

基本信息

批准号：
9754850
负责人：
William Tswenching Pu
金额：
$ 21.01万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-03 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9754850
关键词：
Automation Biology Biomedical Engineering CRISPR/Cas technology Cardiac Cardiac Myocytes Cell Separation Cell Shape Cell membrane Cells Cellular Morphology Cellular Structures Characteristics Computer software Coupling Data Developmental Biology Foundations Genes Genetic Genetic Screening Goals Guide RNA Image Analysis In Vitro Knowledge Label Libraries Longevity Mammalian Cell Mammals Membrane Methodology Methods Microscope Morphology Mosaicism Mus Mutagenesis Organelles Organism Pathway interactions Phenotype Process Property Proteins Protocols documentation Research Role Shapes Sorting - Cell Movement Specific qualifier value Stem cells Structural Protein Structure Subcellular structure System Technology Therapeutic Tissues Tubular formation Tweens Variant Viral Vector base cardiac regeneration cell type design forward genetics gene discovery high throughput technology human disease in vivo insight instrument interdisciplinary collaboration microscopic imaging novel open source programs

项目摘要

Project Summary/Abstract The goal of this project is to develop a platform that will allow cells to be sorted based on properties determined by microscopic imaging. While morphology-based cell cytometers and flow cytometers exist, these instruments do not permit separation of cells by their morphological properties. Combining a “Sorting Microscope” with CRISPR/Cas9-based mutagenesis will enable in vivo or in vitro forward genetic screens to identify genetic programs that regulate single cell phenotypes such as shape, intracellular organization, and protein or organelle subcellular localization. We will perform a proof-of-concept study in which we use the Sorting Microscope to perform a forward genetic screen based on cell morphology. Cardiomyocyte form has evolved to precisely fulfill their functional role. Among these structural adaptations, perhaps the most striking are the transverse tubules (T-tubules), a network of tubular invaginations of the plasma membrane that penetrate into the center of the cardiomyocyte. T-tubules are considered a hallmark of mature CMs and are required for efficient excitation-contraction coupling, yet little is known about the factors that regulate T-tubule formation. Using our platform for in vivo CRISPR/Cas9-based somatic mutagenesis and the Sorting Microscope, we will undertake a proof-of-concept forward genetic screen to identify genes required for T-tubule formation. Our Specific Aim is to establish the technology and methodology to perform forward genetic screens based on cell morphology. In Specific Aim 1.A, we will develop a platform for automated identification, labeling, and sorting of cells by morphology. This open-source hardware and software platform will be designed to facilitate widespread dissemination. In Specific Aim 1.B, we will use the hardware/software platform to identify genes required for T-tubule formation in cardiomyocytes. To achieve these goals, we have assembled an interdisciplinary team consisting of a bioengineering group (Voldman) and a cardiac biology group (Pu). Voldman’s bioengineering group has expertise in image analysis, automation, and in morphology-based cell separation. The Pu lab has expertise in Cas9-based in vivo somatic mutagenesis and in cardiac biology. We anticipate that the resulting technology will enable the power of forward genetics to be unleashed on diverse problems in developmental biology, with direct relevance to human diseases.

项目总结/文摘