Neuronal activation in a transgenic model

• US 20040031065A1
• Filed: 04/25/2003
• Published: 02/12/2004
• Est. Priority Date: 04/26/2002
• Status: Active Grant

• Current Assignee: Carnegie Mellon University, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Carnegie Mellon University, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Barth, Alison L.
• Granted Patent: US 8,952,213 B2
• Time in Patent Office: Days
• US Class Current: 800/3
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/0356 Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The disclosed invention provides compositions and methods for the identification of cells that are functionally activated after stimulation or during an activity while maintaining the viability of the identified cells.

Claims

1. A method to identify one or more cells of a non-human animal, functionally activated in response to a stimulus or during an activity, comprising

introducing, into a cell, or its predecessor, of a non-human animal, a nucleic acid construct comprising one or more regulatory sequences operably linked to a nucleic acid sequence encoding a detectable reporter wherein said regulatory sequence is activated in response to said stimulus or during said activity;

subjecting said animal to said stimulus or inducing said activity in said animal to activate said regulatory sequence(s); and

detecting expression of said reporter in the cells of said animal, while maintaining their viability, to identify one or more cells as functionally activated in response to said stimulus or during said activity.

2. The method of claim 1 wherein said cells are neurons that are involved in brain function.

3. The method of claim 1 wherein said introducing of a nucleic acid construct is by “

knock in”

recombination, insertion of a transgene, or use of a viral vector.

4. The method of claim 3 wherein said introducing of a nucleic acid is via injection into a fertilized cell or blastocyst to produce a transgenic animal.

5. The method of claim 2 wherein said stimulus is administration of a drug, pain, or pleasure;

induction of dendritic outgrowth;

or learning.

6. The method of claim 5 wherein said learning is selected from an elevated plus maze, a light/dark box, an open field test, and a water maze.

7. The method of claim 2 wherein said stimulus is selected from fear;

sensory deprivation or stimulation;

stress;

reward for behavior that they attend to;

conditioning;

visual, audio, location or olfactory cues;

administration of a mind altering drug selected from marijuana, cocaine, heroin, alcohol, or functional equivalents thereof, mating;

aggression;

sexual behavior;

maternal behavior;

social dominance; and

deprivation of water or food; and

said activity is selected from exploratory behavior;

sleep and stages thereof;

narcolepsy;

lactation;

parturition; and

movement through a maze.

8. The method of claim 7 wherein said location cue is linked to drug addiction.

9. The method of claim 8 wherein said drug is selected from nicotine, marijuana, caffeine, cocaine, heroin, and alcohol.

10. The method of claim 1 wherein said regulatory sequences are those of a gene selected from a cellular immediate early gene;

a gene responsive to changes in intracellular Ca++ levels, PKA signaling, cAMP signaling, CAMKIV signaling, and MAPK signaling;

a gene involved in synaptic function;

a gene activated after some patterns of neuronal activity;

prostaglandin endoperoxide H synthase-2 (PGHS-2);

heat shock protein (HSP) 70;

HSP 72;

fosB; and

junB.

11. The method of claim 10 wherein said regulatory sequences are those of a gene selected from c-fos, c-jun, JunD, zif268/krox24/erg1/NGFI-A, krox20/egr2, egr-3, narp, arc/Arg 3.1, beta-activin, homer, cpg-15/neuritin, tyrosine hydroxylase, cox-2, nur77, nurr1, neuronal nitric oxide synthase, brain-derived neurotrophic factor (BDNF), IGF-1, NGFI-B, and trkB.

12. The method of claim 1 wherein said reporter is a luminescent or fluorescent protein.

13. The method of claim 12 wherein said reporter is selected from green fluorescent protein (GFP), modified or enhanced green fluorescent protein (EGFP), yellow fluorescent protein, cyan FP, blue FP, red FP, HcRed, or luciferase.

14. The method of claim 1 wherein said stimulus is physical or chemical.

15. The method of claim 14 wherein said stimulus is electrical.

26. A method to assay cellular physiology of one or more neurons involved in brain function of a non-human animal in response to a stimulus or during an activity comprising

identifying one or more neurons by the method of claim 2;

optionally subjecting said animal to cessation of said stimulus or activity or to a second stimulus or activity; and

detecting cellular physiology of said neuron(s) in situ.

27. The method of claim 26 wherein said detecting is of electrophysiological changes.

16. A method to isolate neurons involved in brain function of a non-human animal in response to a stimulus or during an activity comprising

introducing, into a neuron, or its predecessor, of a non-human animal, a nucleic acid construct comprising one or more regulatory sequences operably linked to a nucleic acid sequence encoding a detectable reporter wherein said regulatory sequence is activated in response to said stimulus or during said activity;

subjecting said animal to said stimulus or inducing said activity in said animal to activate said regulatory sequences;

detecting expression of said reporter in the cells of said animal, while maintaining their viability, to identify one or more neurons as involved in brain function in response to said stimulus or during said activity; and

isolating the identified one or more neurons from said animal.

17. The method of claim 16 wherein said isolating is removal of a slice of brain tissue containing said identified neurons.

18. The method of claim 17 further comprising the step of preparing a gene expression profile from said isolated cells.

19. A method to assay cellular physiology in one or more neurons functionally activated in brain function of a non-human animal in response to a stimulus or during an activity comprising

isolating one or more neurons by the method of claim 16;

contacting said one or more neurons with a test agent or subjecting said one or more neurons to a test growth condition; and

detecting a cellular activity of said neuron(s).

21. The method of claim 19 wherein said contacting is with a candidate drug agent and said cellular physiology comprises a neuronal response to said drug agent.

22. The method of claim 21 wherein said neuronal response is selected from increased firing rate, change in membrane potential, change in kinetics of opening and closing of one or more surface receptor channels, change in number of receptors, and depolarization.

23. The method of claim 19 wherein said cellular physiology comprises dendritic outgrowth or modification of synaptic function.

24. The method of claim 19 wherein said detecting is of electrophysiological changes.

25. The method of claim 24 wherein said changes are selected from changes in dynamic potential, AMPA/NMDA ratios, glutamate receptor subunit composition, miniature EPSC analysis, threshold for action potential generation, short term plasticity paired pulse facilitation, long term potentiation or depression, and sensitivity of plasticity events to pharmacological compounds.

20. The method of claim 20 wherein said isolated neurons are in a section or slice of brain tissue.

28. A transgenic non-human animal the genome of which comprises a first nucleic acid construct comprising one or more first regulatory sequences operably linked to a nucleic acid sequence encoding a first detectable reporter

wherein said regulatory sequence(s) is/are activated in response to a stimulus or during an activity and

wherein said reporter is detectable while maintaining viability of the cell(s) in which it is expressed.

29. The animal of claim 28 wherein said regulatory sequences are those of a gene selected from a cellular immediate early gene;

a gene responsive to changes in intracellular Ca++ levels, PKA signaling, cAMP signaling, CAMKIV signaling, and MAPK signaling;

a gene involved in synaptic function;

a gene activated after some patterns of neuronal activity;

prostaglandin endoperoxide H synthase-2 (PGHS-2);

heat shock protein (HSP) 70;

HSP 72;

fosB; and

junB.

30. The animal of claim 28 wherein said reporter is a luminescent or fluorescent protein.

31. The animal of claim 28 which is a mouse.

32. The animal of claim 28, further comprising a second nucleic acid construct comprising one or more second regulatory sequences operably linked to a nucleic acid sequence encoding a second detectable reporter wherein said second regulatory sequences is/are activated in response to a second stimulus or during a second activity to express said second reporter,

wherein said second sequence, second reporter, and second stimulus or activity are different from said first sequence, first reporter, and first stimulus or activity and

wherein said second reporter is detectable while maintaining viability of the cell(s) in which it is expressed.

33. Progeny of the animal of claim 28, wherein the progeny each have a genome comprising said first nucleic acid construct.

34. Progeny of the animal of claim 32 wherein the progeny each have a genome comprising said first and second nucleic acid constructs.

35. A method of producing the animal of claim 28 comprising introducing said first nucleic acid construct into a fertilized egg, embryo, or blastocyst.

precursor cell (stem cell or blastocyst, fertilized egg)

36. A method of identifying one or more neurons activated by a test compound comprising

administering a test compound to an animal according to claim 28;

identifying neuron(s) expressing said reporter.

37. A method of identifying one or more neurons activated by two different test compounds comprising

administering said test compounds to an animal according to claim 32;

identifying neuron(s) expressing both said first and second reporters.

38. A method of identifying an agent as functionally activating the same neurons as those activated in response to a stimulus or during an activity comprising

providing an animal according to claim 28;

identifying neurons activated in response to a stimulus or during an activity;

cease said stimulus or activity in said animal;

administer an agent to said animal;

identifying neurons activated in response to administration of said agent;

determining whether the neurons identified in response to said stimulus or during said activity are the same as the neurons activated in response to administration of said agent

wherein activation of the same neurons identifies said agent as functionally activating the same neurons as the stimulus or activity.

39. The method of claim 38 wherein said activity is loss of further appetite after eating and said agent is an appetite suppressant.