A new strategy for cell-type specific gene disruption in flies and mice

果蝇和小鼠细胞类型特异性基因破坏的新策略

• 批准号: 9297370
• 负责人: Thomas Robert Clandinin
• 金额: $ 73.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-18 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9297370
• 关键词: Address; Alleles; Alpha Cell; Animal Behavior; Animal Model; Behavior; Behavioral; Behavioral Assay; Benchmarking; Biological Assay; Biological Models; Brain; Calcium; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collection; Communities; Complex; DNA Insertion Elements; DNA Transposable Elements; DNA cassette; Data; Development; Disease; Drosophila genus; Electrical Synapse; Electrophysiology (science); Elements; Enterobacteria phage P1 Cre recombinase; Enzymes; Future; Gated Ion Channel; Gene Expression; Gene Expression Profile; Gene Mutation; Gene Targeting; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomics; Goals; Health; Histologic; Image; Individual; Instinct; Introns; Ion Channel Gating; Label; Lead; Link; Mammals; Measures; Mediating; Membrane Potentials; Molecular; Mus; Mutation; Nervous system structure; Neurodegenerative Disorders; Neurons; Neurosciences; Neurotransmitter Receptor; Neurotransmitters; Output; Pattern; Pharmacology; Physiological; Play; Process; Proteins; Reagent; Reporter; Reporting; Research Personnel; Role; SNAP receptor; Shapes; Specificity; Synapses; Synaptic Transmission; TPH2; Temperature; Transgenic Organisms; Tyrosine 3-Monooxygenase; Validation; Visual; Visual system structure; Work; behavioral study; brain dysfunction; cell type; design; efficacy testing; experience; flexibility; fly; gene function; in vivo; interest; member; nervous system disorder; neural circuit; neural patterning; neuronal circuitry; neuronal excitability; neurophysiology; neuropsychiatric disorder; neuroregulation; neurotransmission; public health relevance; recombinase; relating to nervous system; tool; treatment strategy; two-photon; vesicular GABA transporter; vesicular glutamate transporter 2; virtual; voltage

 DESCRIPTION (provided by applicant): Developing and validating a toolkit for cell type specific gene manipulation PIs: Clandinin and Shah Neurons express complex arrays of genes that play crucial roles in determining neuronal function. As such, single-gene mutations can lead to neurodevelopmental, neurophysiological, and neurodegenerative diseases. However, the nervous system is made up of many different types of neurons, which differ both in the genes they express and the function those genes perform. The ability to inactivate targeted genes only in the cell type of interest is therefore critical for our understanding of neural circuit function This application will generate a generalizable, validated set of transgenic flies and mice for cell type specifically manipulating genes that control the inputs, outputs, and activity patterns of neurons in physiological and behavioral studies of a wide array of circuits. This application describes three goals for developing and validating this toolkit. First, we will generate conditional allelesof 24 genes important for neuronal excitability and signaling in Drosophila. Second, we will validate our cell type specific gene disruption in vivo using a combination of voltage imaging, calcium imaging and behavioral assays, providing strong evidence of the broad utility of this toolkit. Third, since mice are critical model organisms for studying vertebrate nervous systems in health and in disease, we will adapt the same tool we use in flies to mice, targeting a key set of genes controlling excitation, inhibition and neuromodulation. Development of this toolkit will provide th neuroscience community with the means to manipulate essential neural genes with unprecedented precision in cell types of interest, thereby allowing fundamental questions about how genes shape neuronal circuit function to be addressed. As mutations in these classes of genes lead to devastating neurological disorders, this tool will facilitate studies that expand our understanding of these diseases and the treatment possibilities. As pharmacological approaches to treating brain dysfunction are ultimately limited by molecular specificity, understanding cell-type specific gene function is critical to the development of new treatment strategies. Finally, as the tool we will develop can be generalized to virtually any gene, future studies can extend the use of this tool to any gene of interest, in either flies or mice.

 描述（由申请人提供）：开发和验证用于细胞类型特异性基因操作PI的工具包：Clandinin和Shah神经元表达在决定神经元功能中起关键作用的复杂基因阵列。因此，单基因突变可导致神经发育、神经生理和神经退行性疾病。然而，神经系统是由许多不同类型的神经元组成的，它们表达的基因和这些基因执行的功能都不同。因此，仅在感兴趣的细胞类型中检测靶基因的能力对于我们理解神经回路功能至关重要。 特别是操纵基因，控制输入，输出，和神经元的活动模式在生理和行为研究的广泛的电路。这个应用程序描述了开发和验证这个工具包的三个目标。首先，我们将在果蝇中产生对神经元兴奋性和信号传导重要的24个基因的条件等位基因。其次，我们将使用电压成像，钙成像和行为测定的组合来验证我们的细胞类型特异性基因破坏，为该工具包的广泛实用性提供强有力的证据。第三，由于小鼠是研究健康和疾病中的脊椎动物神经系统的关键模型生物，我们将采用我们在苍蝇中使用的相同工具，针对控制兴奋，抑制和神经调节的关键基因组。该工具包的开发将为神经科学界提供在感兴趣的细胞类型中以前所未有的精度操纵必需神经基因的方法，从而允许解决有关基因如何塑造神经元回路功能的基本问题。由于这类基因的突变会导致破坏性的神经系统疾病，因此该工具将有助于扩大我们的研究范围。 了解这些疾病和治疗的可能性。由于治疗脑功能障碍的药理学方法最终受到分子特异性的限制，因此了解细胞类型特异性基因功能对于开发新的治疗策略至关重要。最后，由于我们将开发的工具可以推广到几乎任何基因，未来的研究可以将该工具的使用扩展到任何感兴趣的基因，无论是苍蝇还是老鼠。