Light-Induced Genetic Alterations within Single Cell of a Live Vertebrate Animal

光诱导活体脊椎动物单细胞内的遗传改变

基本信息

批准号：
8929327
负责人：
John M Parant
金额：
$ 18.38万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-30 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8929327
关键词：
Ablation Address Alleles Animal Disease Models Animal Model Animals Architecture Area Biochemical Biological Assay Biological Markers Biological Models Biomedical Research Cancer Biology Cell Culture System Cell Survival Cells Chromatin Complementary DNA Complex Data Set Defect Developmental Biology Diabetes Mellitus Disease Disease Progression Drosophila genus Eating Embryo Environment Gene Expression Profile Gene Mutation Genes Genetic Genetic Crosses Genetic Recombination Genome engineering Goals Guide RNA Health Hormones Image Immunology In Situ In Vitro Individual Injection of therapeutic agent Knock-out Life Light Light Cell Measures Messenger RNA Methods Modeling Mus Mutation Nerve Degeneration Neurobiology Organism Population Proteins Proteome Research Research Personnel System Temperature Testing Time Tissues Transgenic Animals Transgenic Organisms Transplantation Vertebrates Whole Organism Zebrafish base biological systems cDNA Expression cancer cell cell type disease phenotype disorder prevention early onset egg genetic manipulation human disease in vivo innovation interest knockout gene loss of function optogenetics promoter recombinase single cell analysis spatiotemporal temporal measurement tool tumor

项目摘要

DESCRIPTION: Animal modeling of disease states and bioreporters for in situ analysis of cellular dynamics are an essential aspect of biomedical research; however, most studies focus on genetic manipulation within the whole organism or tissue of interest, which does not accurately reflect disease states that are often associated with defects in individual cells or groups of cells. This discrepancy is largely due to the technical challenges of single cell manipulation and analysis within an intact organism. While ongoing SCAP studies are characterizing the transcriptome, proteome, and chromatin alterations within single cells, a biological system to test the impact of these single cell alterations is necessary. Therefore, consistent with this FOA, we believe that a system for con- trolled genetic manipulation (resulting in loss of function alleles or expression of exogenous cDNAs/bioreporters) within a single cell of a viable organism is required. This system should not alter or perturb the surrounding environment and should allow for potential systems based analysis and biomarker incorporation. Therefore we hypothesize that: 1) through the use of optogenetic (light inducible) Cre transgenic animals, single cell Cre recombination can be achieved allowing for single cell inducible gene alterations (i.e. conditional knockout or bioreporter/gene of interest expression); and 2) through the use of a Cre inducible Cas9 system, single and multiple gene ablations can be rapidly achieved within an individual cell of an intact organism. These two systems will be established within the zebrafish embryo but can be applied to all model organisms. Upon completion of this proposal we will have established versatile and efficient single cell tools for selective genetic alterations that will facilitate single cell analysis in a multitude of research fields. We anticipate that this research will have a broad positive impact on a number of other human diseases including (but not limited to) neurobiology, immunology, cancer biology, and developmental biology. This proposal is in alignment with the SCAP and this FOA objectives: 1) to develop tools that "minimize cell perturbation and permit viability of cells for repeated measures over time"; 2) "Systems-level single cell dataset analysis or modeling... in the context of tissues or whole organisms; and 3) "the discovery of new, innovative tools for spati- otemporal imaging, manipulation, analysis and modeling of a biologically relevant population of cells with minimal perturbation".

描述：疾病状态的动物模型和用于细胞动力学原位分析的生物报告器是生物医学研究的一个重要方面，然而，大多数研究侧重于整个有机体或感兴趣组织内的基因操作，这并不能准确反映疾病状态。这种差异通常与单个细胞或细胞群的缺陷有关，这主要是由于完整生物体中单细胞操作和分析的技术挑战，而正在进行的 SCAP 研究正在表征单个细胞内的转录组、蛋白质组和染色质改变。细胞，测试这些单细胞改变的影响的生物系统是必要的，因此，与此 FOA 一致，我们认为在一个细胞内进行受控遗传操作（导致功能等位基因丧失或外源 cDNA/生物报告基因的表达）的系统是必要的。该系统不应改变或扰乱周围环境，并且应允许基于潜在系统的分析和生物标志物整合，因此我们认为：1）通过使用光遗传学（光）。诱导型）Cre 转基因动物，可以实现单细胞 Cre 重组，从而允许单细胞诱导型基因改变（即条件性敲除或生物报告基因/感兴趣的基因表达）；以及 2）通过使用 Cre 诱导型 Cas9 系统、单基因和多基因消融可以在完整生物体的单个细胞内快速实现，这两个系统将在斑马鱼胚胎内建立，但可以应用于所有模型生物体。用于选择性遗传改变的有效单细胞工具，将促进多个研究领域的单细胞分析，我们预计这项研究将对许多其他人类疾病产生广泛的积极影响，包括（但不限于）神经生物学、免疫学、该提案与 SCAP 和 FOA 的目标一致：1）开发“最小化细胞扰动并允许细胞随时间重复测量的活力”的工具；2）“系统级单细胞”。数据集分析或建模...在组织或整个生物体的背景下；3）“发现新的、创新的工具，以最小的扰动对生物相关的细胞群进行时空成像、操作、分析和建模”。