We propose to develop a genetic mosaic system in mice that allows simultaneous labeling and genetic manipulation of defined neuronal populations, down to the level of single isolated neurons in vivo. This system, which we have named """"""""MADM"""""""" (for Mosaic Analysis with Double Markers), utilizes two hybrid marker genes knocked-in at identical locations on homologous chromosomes. Each marker gene is interrupted by a loxP-containing intron and neither expresses a functional protein. Only upon Cre-mediated recombination between the two loxP sites on the homologous chromosomes are functional marker genes restored. Depending on the cell-cycle stage at which recombination takes place and the segregation pattern of the chromosomes after recombination, daughter cells are labeled with one or both markers. We have preliminary results indicating that MADM can be used to generate, with high efficiency, inter-chromosomal exchanges in both postmitotic neurons and in dividing neural precursors. By further developing this method and its variations we will be able to label defined neuronal populations and single neurons with genetically encoded markers, in live or fixed brains. It will also be possible to create genetic mosaics such that cells expressing the first functional marker are homozygous mutant for a gene of interest, whereas cells expressing the second functional marker are homozygous wild type, while the rest of the animal is heterozygous. MADM will allow investigation of the relationship between cell lineage and neural circuits during development, tracing of neural circuits in the adult nervous system, and conditional knock-out of candidate genes of interest as well as overexpression of transgenes in single isolated neurons. This method can also be used to create mouse models for human diseases such as loss of heterozygosity in cancer and neurological diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS050835-01
Application #
6858492
Study Section
Special Emphasis Panel (ZRG1-MDCN-B (55))
Program Officer
Mamounas, Laura
Project Start
2005-01-01
Project End
2008-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
1
Fiscal Year
2005
Total Cost
$365,687
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Luo, Liqun; Callaway, Edward M; Svoboda, Karel (2018) Genetic Dissection of Neural Circuits: A Decade of Progress. Neuron 98:865
Luo, Liqun; Callaway, Edward M; Svoboda, Karel (2018) Genetic Dissection of Neural Circuits: A Decade of Progress. Neuron 98:256-281
Beier, Kevin T; Kim, Christina K; Hoerbelt, Paul et al. (2017) Rabies screen reveals GPe control of cocaine-triggered plasticity. Nature 549:345-350
Lasrado, Reena; Boesmans, Werend; Kleinjung, Jens et al. (2017) Lineage-dependent spatial and functional organization of the mammalian enteric nervous system. Science 356:722-726
Allen, William E; DeNardo, Laura A; Chen, Michael Z et al. (2017) Thirst-associated preoptic neurons encode an aversive motivational drive. Science 357:1149-1155
DeNardo, Laura; Luo, Liqun (2017) Genetic strategies to access activated neurons. Curr Opin Neurobiol 45:121-129
Fran├žois, Amaury; Low, Sarah A; Sypek, Elizabeth I et al. (2017) A Brainstem-Spinal Cord Inhibitory Circuit for Mechanical Pain Modulation by GABA and Enkephalins. Neuron 93:822-839.e6
Wagner, Mark J; Kim, Tony Hyun; Savall, Joan et al. (2017) Cerebellar granule cells encode the expectation of reward. Nature 544:96-100
Huang, Z Josh; Luo, Liqun (2015) NEUROSCIENCE. It takes the world to understand the brain. Science 350:42-4
DeNardo, Laura A; Berns, Dominic S; DeLoach, Katherine et al. (2015) Connectivity of mouse somatosensory and prefrontal cortex examined with trans-synaptic tracing. Nat Neurosci 18:1687-1697

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