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

 产品说明：疾病状态的动物模型和用于原位分析细胞动力学的生物报告是生物医学研究的一个重要方面;然而，大多数研究都集中在整个生物体或感兴趣的组织内的遗传操作，这并不能准确地反映疾病状态，这些疾病状态通常与单个细胞或细胞组中的缺陷有关。这种差异在很大程度上是由于在完整生物体内进行单细胞操作和分析的技术挑战。虽然正在进行的SCAP研究正在表征单细胞内的转录组、蛋白质组和染色质改变，但测试这些单细胞改变的影响的生物系统是必要的。因此，与该FOA一致，我们认为需要在活生物体的单细胞内进行受控遗传操作（导致功能等位基因的丧失或外源cDNA/生物报告基因的表达）的系统。该系统不应改变或干扰周围环境，并应允许基于潜在系统的分析和生物标志物掺入。因此，我们假设：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，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）;和2）通过使用Cre诱导型Cas9系统，可以在完整生物体的单个细胞内快速实现单个和多个基因消融。这两个系统将在斑马鱼胚胎中建立，但可以应用于所有模式生物。在完成这项提案后，我们将建立多功能和有效的单细胞工具，用于选择性遗传改变，这将有助于在众多研究领域进行单细胞分析。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，癌症生物学和发育生物学。该提案符合SCAP和FOA的目标：1）开发“最大限度地减少细胞扰动并允许细胞活力随时间重复测量”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情况下;以及3）“发现新的、创新的工具，用于以最小的扰动对生物学相关的细胞群进行时空成像、操作、分析和建模”。

 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".