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.
Public Health Relevance Statement: We propose to develop an efficient and inexpensive toolkit for ablating or reversibly inactivating specific cell types in the brain or other organs of mammals This innovative research toolkit will facilitate research and drug discovery in the neurological and metal health areas.
NIH Spending Category: Biotechnology; Gene Therapy; Genetics; Neurosciences
Project Terms: Ablation; adeno-associated viral vector; Allatostatin; Antibodies; Area; base; Biological; Brain; cell type; Cells; Chimeric Proteins; Communities; Complex; cost; Cytomegalovirus; Cytotoxin; Data; DNA; DNA Sequence; Dose; Drosophila allatostatin receptor; drug discovery; enhanced green fluorescent protein; Equipment; Gene Expression; Genes; Genetic Recombination; Genetic Transcription; Goals; Health; Informatics; Injection of therapeutic agent; innovation; interest; Label; Laboratories; Ligands; Link; Mammalian Cell; Mammals; Metals; Methods; Mus; neural circuit; Neurologic; Neurons; Nitroreductases; optogenetics; Organ; Phenotype; Physiological; Prodrugs; Promotor (Genetics); Proteins; Protocols documentation; Publishing; receptor; recombinase; Research; Research Personnel; RNA Splicing; Site; Techniques; Technology; Testing; Time Study; Transcription Initiation; Transgenic Animals; Transgenic Mice; vector
