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Tattletales

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Tattletales And T-Bow Update 20140602mon, George Mcnamara Jun 2014

Tattletales And T-Bow Update 20140602mon, George Mcnamara

George McNamara

Tattletales and T-Bow Update 20140602Mon

http://0-works.bepress.com.library.simmons.edu/gmcnamara/42

Please see also http://0-works.bepress.com.library.simmons.edu/gmcnamara/26

Tattletales: multiplex fluorescent protein biosensors by spatial localization with TALE-FPs, Cas9-FPs, ZF-FPs, LacI-FPs, TetR-FPs, etc.

T-Bow: Rainbow T-cells and Tumor cells (and ES cells, iPS cells, other cells and organisms). You can think of this as "Brainbow meets TALENs/Cas9/ZFNs/other DNA sequence specific binding proteins".

If not familiar with Brainbow, see

http://en.wikipedia.org/wiki/Brainbow

If not familiar with TALENs, Cas9, etc, see

http://www.addgene.org/genome_engineering/

Big idea: localizing fluorescent proteins - and/or Nano-Lanterns ...


Pubspectra Tattletales, George Mcnamara Feb 2013

Pubspectra Tattletales, George Mcnamara

George McNamara

Tattletales for Multiplex Fluorescent Reporters in Single Cells for Metabolomics

George McNamara

As of April 2013: L.J.N. Cooper & D.A. Lee Cellular Immunotherapy Lab, University of Texas M.D. Anderson Cancer Center, Houston, TX

Email: gtmcnamara@mdanderson.org, geomcnamara@earthlink.net

Tattletales is my concept for spatial multiplexing many fluorescent protein (FP) biosensors in the same live cell. For example, there are excellent FP biosensors to Ca++ ions, pH, glucose, ribose, glutamine, glutamate, ATP, redox, ROS, pyruvate, cAMP, cGMP, IP3, PI(3,4,5)P3, cell cycle indicators (Fucci2), PKA, PKC, photsphatases, caspase(s) [1, 2]. However, these are typically used one biosensor per experiment, due in part to flooding the cell with soluble biosensor. That is, conventionally, either a metabolite (glucose) reporter or a signal transduction (Ca++) reporter can be imaged. By flooding the cell with the reporter, signal to noise ratio is compromized by autofluorescence.

Tattletales takes advantage of spatial multiplexing to both increase the number of different reporters, and improve signal to noise ratio by localizing each biosensor to a small volume. I started with the observation by Robinett et al [3] who localized 512 GFP-nls-LacI to a 256 LacO array as a 200 nm diameter diffraction limited spot (nuclear background due to overexpression). Many thousands of DNA binding proteins, of known sequence specifities, exist (LacI, TetR, GalR, etc for cell line studies; ZF-FPs, TALE-FPs to STRs, telomere repeat binding factors-FPs, etc for primary cells) and can be fused (as cDNAs) to different fluorescent proteins and FP biosensors.

Many biosensors are available as affinity series [1, 4], now enabling extended dynamic range. I realized that spatial multiplexing of many DNA binding protein-reporters by localizing to different spots in the cell nucleus and distinguished by combinatorial addressing, where N address colors provide 2^N addresses (example, 3 colors is 2^3 = 8 combinations). Multiplexing ...