AAV-based toolkit for targeting specific cell types

基于 AAV 的工具包，用于针对特定细胞类型

• 批准号: 8481519
• 负责人: DMITRY GERASHCHENKO
• 金额: $ 32.75万
• 依托单位: NEUROTARGETING SYSTEMS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8481519
• 关键词: Ablation; Allatostatin; Antibodies; Area; Biological; Brain; Cells; Chimeric Proteins; Communities; Complex; Cytomegalovirus; Cytotoxin; DNA; DNA Sequence; Data; Dose; Drosophila allatostatin receptor; Equipment; Gene Expression; Genes; Genetic Recombination; Genetic Transcription; Goals; Health; Informatics; Injection of therapeutic agent; Label; Laboratories; Ligands; Link; Mammalian Cell; Mammals; Metals; Methods; Mus; Neurologic; Neurons; Nitroreductases; Organ; Phenotype; Physiological; Prodrugs; Proteins; Protocols documentation; Publishing; RNA Splicing; Research; Research Personnel; Site; Techniques; Technology; Testing; Time Study; Transcription Initiation; Transgenic Animals; Transgenic Mice; adeno-associated viral vector; base; cell type; cost; drug discovery; enhanced green fluorescent protein; innovation; interest; neural circuit; optogenetics; promoter; receptor; recombinase; vector

DESCRIPTION (provided by applicant): Our goal is to develop an efficient and inexpensive toolkit for ablating or reversibly inactivating specific cell types in the brain or other organs of mammals. We propose new techniques in which the genes for nitroreductase (NTR) or Drosophila allatostatin receptor (AlstR) are delivered into cells using an adeno-associated viral vector (AAV). Both are fusion proteins linked to enhanced green fluorescent protein (EGFP), making it easy to identify transfected cells. The transfected cells are then either ablated by an i.p. injection of CB1954 or inactivated by a local injection of allatostatin. These new methods provide several advantages over related techniques such as optogenetics or ablation of cells using cytotoxins (e.g. saporin) conjugated to antibodies or ligands. These benefits include a high selectivity of cell targeting, a relatively low cost for the kit's components, and no requirement for expensive equipment. Another advantage is that AAVs efficiently transfect mammalian cells and can be used under BSL1, the biosafety level approved in most biological laboratories. With these advantages, we expect that our toolkit will be used in many labs to efficiently test the physiological functions of different cell types. To achieve high selectivity f cell targeting, these techniques use transgenic mice expressing Cre recombinase under control of cell-specific promoters. Cre-Lox recombination involves the targeting of specific sequences of DNA that are flanked by loxP sites. This recombination can splice out or invert the DNA fragment between the loxP sites. In our proposed studies, Cre recombinase will either splice out a STOP cassette or invert the insert sequence into the sense direction, allowing the expression of foreign genes only in targeted cells. The Specific Aims of the proposed project are to: 1. Construct AAV vectors that enable robust expression of nitroreductase and allatostatin receptor selectively in cells containing Cre recombinase. 2. Test the constructed AAV vectors in several lines of Cre-expressing mice and identify the most efficient protocol for ablation or inactivation f the Cre-containing cells. We believe that our toolkit will be of great help to the research community due to the unique and powerful features of the method.

描述（由申请人提供）：我们的目标是开发一种高效且廉价的工具，用于消融或可逆地灭活大脑或其他器官中的特定细胞类型