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MULTICOLOR CELL MEMBRANE PROFILING FOR DEVELOPMENT AND DISEASE

Award Number: R43GM108470
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/01/2013
PERIOD OF PERFORMANCE END DATE: 05/31/2015

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MULTICOLOR CELL MEMBRANE PROFILING FOR DEVELOPMENT AND DISEASE - DESCRIPTION: Tracing neuronal connections with lipophilic carbocyanine dyes has revolutionized neuroanatomical tract tracing and is an essential feature to understand development of brain connections in both control and mutant mice in particular since multiple colors of dyes can be used. More recently, lipophilic dyes have also gained ground in labeling blood vessels by directly staining the endothelial cell membranes upon contact. A persistent problem that blocks even wider use of these extremely successful dyes is their limited combination with other procedures such as immunostaining or detailed histology that requires dehydration for embedding, since the dye molecules are not permanently bound to the membranes and can either leak out or be easily washed away by lipophilic solvents or detergents. Attempts to overcome these problems have thus far been at best partially successful. Therefore, to broaden even further the use of carbocyanine lipophilic dyes we propose to develop fixable carbocyanine dyes that can be bonded to lysine groups in membrane bound proteins thereby retaining the dyes in the membranes even after the lipid bilayers has been removed with detergents or organic solvents. We plan to optimize the use of these dyes by developing a protocol that allows combination of multiple distinct fluorophores to maximize the information gained from a given model organism. Specifically, our Specific Aims are: (1) Synthesize four spectrally distinct fixable lipophilic dyes, three for nerve tract tracing and one or blood vessel labeling, that are compatible with standard fixation techniques used in tissue processing and immunocytochemistry protocols. Dyes synthesized will feature an aromatic N-hydroxysuccinimide ester group to provide covalent anchoring to membrane proteins. (2) Evaluate fixable dyes in standardized test systems in fixed tissue using processing techniques needed for high resolution histology at the light microscope level. In this aim we will characteriz how long these dyes are retained in tissue after treatment with organic solvents and detergents and how washing out progresses over time. We will also characterize the conditions under which these dyes can be best combined with each other and with immunochemistry and/or staining for dying cells. (3) Create test products to market each dye alone or in combination to the research community. We will develop test products to be sent to 5-10 collaborators in the neuroscience community along with the published protocol developed in SA2. Combined, these three aims will provide novel reagents useful for studies related to breaches in the blood-brain barrier such as tumors, trauma and neurodegenerative diseases as well as normal development.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2013 ( Subtotal = $219,935 )
2013	2013	MOLECULAR TARGETING TECHNOLOGIES INC	833 LINCOLN AVE UNIT 9	WEST CHESTER	PA	19380	CHESTER	USA	Biomedical Research and Research Training	000	1	8/5/2013	NEW	$219,935
														Subtotal = $219,935

Grand Total All Awards = $219,935

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MULTICOLOR CELL MEMBRANE PROFILING FOR DEVELOPMENT AND DISEASE

Award Number: R43GM108470

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/01/2013

PERIOD OF PERFORMANCE END DATE: 05/31/2015

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