EP1888757A2 - Methodes et produits permettant de determiner l'expression du gene f4/80 dans des cellules microgliales - Google Patents

Methodes et produits permettant de determiner l'expression du gene f4/80 dans des cellules microgliales

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Publication number
EP1888757A2
EP1888757A2 EP06759424A EP06759424A EP1888757A2 EP 1888757 A2 EP1888757 A2 EP 1888757A2 EP 06759424 A EP06759424 A EP 06759424A EP 06759424 A EP06759424 A EP 06759424A EP 1888757 A2 EP1888757 A2 EP 1888757A2
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EP
European Patent Office
Prior art keywords
disease
cell
condition
expression
transgenic animal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06759424A
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German (de)
English (en)
Inventor
Jane Relton
Colleen M. Mullen
Catherine A. Hession
Martin Scott
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biogen Inc
Biogen MA Inc
Original Assignee
Biogen Idec Inc
Biogen Idec MA Inc
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Filing date
Publication date
Application filed by Biogen Idec Inc, Biogen Idec MA Inc filed Critical Biogen Idec Inc
Publication of EP1888757A2 publication Critical patent/EP1888757A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the invention relates in some aspects to methods, products, and kits for evaluating and monitoring microglial cell activation in cells, tissues, and animals and identifying compounds that enhance or inhibit microglial cell activation.
  • Microglial cells are of hematopoietic origin and function as the innate immune cells of the brain and spinal cord. These cells form a ramified network throughout the central nervous system (CNS) and are thought to be important in maintaining the CNS in an immune- privileged state.
  • CNS central nervous system
  • neurodegenerative conditions such as Parkinson's disease (Orr et al, 2002, Progress In Neurobiology 68:325-340, Liu & Hong, 2003, J. Pharmacol Exp. Ther. 304(l):l-7), Alzheimer's disease (Akiyama et al, 2000, Alzheimer Dis Assoc. Disord.
  • neuropathic pain Tsuda et al, 2005, Trends in Neurosciences 28(2): 101-107
  • amyotrophic lateral sclerosis ALS
  • microglia are known to become activated and prevailing opinion suggests they actually contribute to the pathogenesis of these and many other neurological diseases by exacerbating the underlying pathology and in some instances causing neuronal cell death.
  • microglial cells to exacerbate neurodegeneration is thought to be due, at least in part, to their ability to generate and secrete pro-inflammatory molecules such as tumor necrosis factor- ⁇ , interleukin-l ⁇ , interleukin-6 and nitric oxide (Hanisch, 2002, GHa 40:140-155; John et al, 2003, The Neuroscientist 9(1): 10-22).
  • pro-inflammatory molecules such as tumor necrosis factor- ⁇ , interleukin-l ⁇ , interleukin-6 and nitric oxide
  • aspects of the invention relate to constructs, transgenic cells, transgenic cell lines, and transgenic non-human animals that are useful to detect and monitor macrophage (e.g., microglial) activation.
  • the invention includes, in part, methods of monitoring and assessing microglial activation using a construct with inducible expression.
  • Cells transfected with a genetic reporter construct can be used in assays of macrophage (e.g., microglial) activation.
  • Reporter constructs of the invention can also be used to generate non-human transgenic animals.
  • constructs, transgenic cells, and transgenic animals of the invention may include a detectable reporter protein and are designed to express a detectable reporter protein (e.g., GFP or EGFP, or luciferase, etc.) under the direction of a macrophage- specific promoter, (for example, an F4/80 promoter or fragment thereof).
  • a detectable reporter protein e.g., GFP or EGFP, or luciferase, etc.
  • a macrophage- specific promoter for example, an F4/80 promoter or fragment thereof.
  • expression of the detectable reporter is increased in the central nervous system and in other macrophage-associated tissues after inflammatory challenge or nerve injury.
  • the amount and location of expression of the detectable reporter can be determined using any suitable detection method.
  • the amount of reporter protein expression may be proportional to the extent of microglial activation, thus allowing determination of the activation state and level of activation of microglial cells in the transgenic animal.
  • macrophage e.g., microglial activation can be assessed in the brain and/or spinal cord.
  • transgenic mice have been generated that express reporter proteins enhanced green fluorescent protein (EGFP) and firefly luciferase (Luc) under the direction of a fragment of the F4/80 promoter
  • the invention includes methods of monitoring and assessing microglial activation using cell and animal models
  • use of luciferase as a reporter protein in a transgenic animal of the invention allows a real-time assessment of microglial activation in the animal.
  • non-human transgenic animals are provided.
  • the non-human transgenic animals include somatic cells that contain a genomic reporter transgene, wherein expression of the transgene is inducible in the central nervous system, and wherein an expression product of the reporter transgene is a detectable expression product, hi some embodiments, the animal includes more than one copy of a transgene. In some embodiments, the animal includes at least two different transgenes. In certain embodiments, the animal includes multiple copies of a transgene. Li some embodiments, the non-human transgenic animal also includes germ cells that contain the genomic reporter transgene. In some embodiments, expression of the transgene is inducible in microglial cells and tissue macrophages. In some embodiments, the animal is a mouse.
  • the non- human transgenic animal also includes one or more genomic mutations associated with a neurological disease or disorder.
  • the reporter transgene is operatively linked to a fragment of an F4/80 promoter, and wherein the expression of the detectable expression product of the reporter transgene is under the control of the fragment of the F4/80 promoter.
  • expression of more than one detectable expression product of the reporter transgene is under the control of the fragment of the F4/80 promoter.
  • the detectable expression product of the reporter transgene comprises a bioluminescent product or a fluorescent product, hi some embodiments, the detectable expression product of the reporter transgene comprises a bioluminescent product.
  • the bioluminescent product is a luciferase.
  • the luciferase is a firefly luciferase, Renilla luciferase, or a Genji-botaru luciferase.
  • the nucleotide sequence of the fragment of the F4/80 promoter comprises the nucleic acid sequence set forth as SEQ ID NO:2.
  • an animal that is a descendent of any of non-human transgenic animals of any of the foregoing embodiments or aspects of the invention is provided.
  • the method includes determining a level of expression of the detectable expression product in a biomolecular pathway in the animal with the neurological disease or condition and comparing the level of expression in the biomolecular pathway to a control level of expression of the detectable expression product in the biomolecular pathway, wherein a difference in the level of expression of the detectable expression product in the non-human transgenic animal compared to the control level of the detectable expression product in the biomolecular pathway identifies the biomolecular pathway as associated with a the neurological disease or condition in the animal, hi certain embodiments, the disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, peripheral a neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy, peripheral neuropathy,
  • the transgenic animal has the neurological disease, or condition. In some embodiments, the transgenic animal is a model for the neurological disease or condition. According to some aspects of the invention, methods of identifying a candidate compound as modulating activity of a biomolecular pathway associated with a neurological disease or condition in a transgenic animal of any of the foregoing embodiments or aspects of the invention are provided.
  • the methods include determining the level of expression of the detectable expression product in the transgenic animal as a measure of the activity of a biomolecular pathway associated with a neurological disease or condition in the animal, administering to the transgenic animal a candidate compound, determining a subsequent level of expression of the detectable expression product in the transgenic animal after administration of the candidate compound, and comparing the level of expression of the detectable expression product in the transgenic animal before the administration of the candidate compound, to the level of expression of the detectable expression product in the transgenic animal after the administration of the candidate compound, wherein a difference in the levels identifies the candidate compound as modulating activity of the biomolecular pathway associated with the neurological disease or condition.
  • the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, or aging.
  • the transgenic animal has the neurological disease or condition.
  • the transgenic animal is a model for the neurological disease or condition. According to yet another aspect of the invention, methods of monitoring the onset, progression, and/or regression of a neurological disease or condition in a non-human transgenic animal of any of the foregoing aspects or embodiments of the invention are provided.
  • the methods include determining a level of expression of the detectable expression product in the animal and comparing the level of expression to a control level of expression as an indication of the onset, progression, or regression of the neurological disease or condition in the transgenic animal, ha certain embodiments, the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, or aging.
  • the transgenic animal has the neurological disease or condition.
  • the transgenic animal is a model for the neurological disease or condition.
  • the methods include administering to the transgenic animal a candidate therapeutic compound or treatment regimen, determining a level of expression of the detectable expression product in the transgenic animal administered the candidate therapeutic compound or treatment regimen, comparing the level of expression of the detectable expression product in the transgenic animal administered the candidate therapeutic compound or treatment regimen, to a control level of expression of the detectable expression product, wherein a difference in the levels indicates the efficacy of the candidate therapeutic compound or treatment regimen for the neurological disease or condition, hi some embodiments, the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, or aging, hi
  • a nucleic acid construct includes a reporter gene operatively linked to a fragment of an F4/80 promoter wherein the reporter gene encodes a detectable expression product, and wherein the expression of the detectable expression product of the reporter gene is under the control of the fragment of the F4/80 promoter, hi some embodiments, the nucleotide sequence of the fragment of the F4/80 promoter comprises the nucleic acid sequence set forth as SEQ ID NO:2.
  • the detectable expression product of the reporter gene comprises a bioluminescent product or a fluorescent product, hi some embodiments, the detectable expression product of the reporter gene comprises a luciferase.
  • the luciferase is firefly luciferase, Renilla luciferase, or a Genji-botaru luciferase.
  • the reporter is GFP, j3-galactosidase, or chloramphenicol acetyltransferase gene (CAT).
  • cells are provided.
  • the cells includes a nucleic acid construct of any of the foregoing aspects or embodiments of the invention.
  • the cell also includes one or more genomic mutations associated with a neurological disease or condition.
  • expression of more than one detectable expression product of the reporter gene is under the control of the fragment of the F4/80 promoter.
  • the cell is derived from a healthy or pathological microglial or macrophage tissue or cell.
  • cell lines are provided.
  • the cell lines includes a nucleic acid construct of any of the foregoing aspects or embodiments of the invention.
  • the cell line also includes one or more genomic mutations associated with a neurological disease or condition.
  • expression of more than one detectable expression product of the reporter gene is under the control of the fragment of the F4/80 promoter.
  • the cells are derived from healthy or pathological microglial or macrophage tissues or cells.
  • methods that include performing a cellular screening assay with a cell or a cell line of any of the foregoing aspects or embodiments of the invention are provided.
  • methods that include using a cell of any of the foregoing aspects or embodiments for the in vitro formation of tissue are provided.
  • methods for testing whether a candidate compound induces expression of the reporter in a cell or cell line of any of the foregoing aspects of embodiments are provided. The methods include contacting the cell or cell line with the compound to be tested and comparing the expression of the reporter to a control.
  • the cells are mammalian cells.
  • the mammalian cells are mouse cells.
  • the mammalian cells are human cells.
  • the reporter is firefly luciferase, Renilla luciferase, or a Genji- botaru luciferase; and wherein the promoter is a fragment of an F4/80 promoter.
  • the fragment of the F4/80 promoter comprises the nucleotide sequence set forth as SEQ ID NO:2.
  • the methods include determining the level of expression of the detectable expression product in the cell or cell line as a measure of the activity of a biomolecular pathway associated with a neurological disease or condition in the cell or cell line, contacting the cell or cell line with the candidate compound, determining a subsequent level of expression of the detectable expression product in the cell or cell line after contact with the candidate compound, comparing the level of expression of the detectable expression product in the cell or tissue before contact with the candidate compound, to the level of expression of the detectable expression product in the cell or cell line after the contact of the candidate compound, wherein a difference in the levels identifies the candidate compound as modulating activity of the biomolecular pathway associated with the neurological disease or condition.
  • the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, or aging, hi certain embodiments, the cell or cell line has the neurological disease of condition. In some embodiments, the cell or cell line is a model for the neurological disease or condition.
  • ALS amyotrophic lateral sclerosis
  • methods for monitoring the onset, progression, and/or regression of a neurological disease or condition in a cell or cell line of any of the foregoing aspects or embodiments are provided.
  • the methods include determining a level of expression of the detectable expression product in the cell or cell line and comparing the level of expression to a control level of expression as an indication of the onset, progression, or regression of the neurological disease or condition in the cell or cell line.
  • the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, , spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy or peripheral nerve injury, a psychiatric disease.
  • the cell or cell line has the neurological disease or condition, hi some embodiments, the cell or cell line is a model for the neurological disease or condition.
  • methods for evaluating the efficacy of a candidate therapeutic compound or treatment regimen for a neurological disease or condition in a cell or cell line of any of the foregoing aspects or embodiments are provided.
  • the methods include contacting the cell or cell line with a candidate therapeutic compound or treatment regimen, determining a level of expression of the detectable expression product in the cell or cell line contacted with the candidate therapeutic compound or treatment regimen, comparing the level of expression of the detectable expression product in the cell or cell line contacted with the candidate therapeutic compound or treatment regimen, to a control level of expression of the detectable expression product, wherein a difference in the levels indicates the efficacy of the candidate therapeutic compound or treatment regimen for the neurological disease or condition.
  • the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, or aging, hi some embodiments, the cell or cell line has the neurological disease or condition. In some embodiments, the cell or cell line is a model for the neurological disease or condition.
  • ALS amyotrophic lateral sclerosis
  • Fig. 1 shows a diagram of a F4/80GFP//we reporter construct generated for transgenesis
  • Fig. 2. provides two graphs indicating that peripheral injection of LPS (5mg/kg IP) resulted in increased expression of luciferase.
  • Fig. 2A shows level of fluorescence after various time periods following injection.
  • the numbers 34, 36, 39, and 41 represent numbers assigned to individual F480-GFP/Luc transgenic mice that have been injected intraperitoneally with LPS.
  • Fig. 2 B shows a curve the mean values of bioluminescence for these four mice over the various time points measured +/- standard error of the mean.
  • Fig. 3. provides a graph indicating microglial activation in the spinal cord of F4/80 GFP/Xwc mice.
  • the graph indicates that luciferase activity increased in the dorsal horn of the spinal cord in a time-dependent manner as compared to sham control animals.
  • Control animals were not injected with LPS or PBS but were injected only with luciferin on days 0 (baseline), 1, 3, 6, 8, 10, and 13.
  • Fig. 4. is a graph demonstrating pharmacological inhibition of LPS-induced luciferase expression in the F4/80 GFP/£wc mouse. The average radiance of each animal was detected at various intervals over a 78 hour time span after injections. The graph indicates a rise and fall of radiance in the LPS (top trace with circles) and minocycline (second highest trace, open squares) injected animals.
  • Fig. 5 is a graph indicating biochemical measurement of luciferase activity in tissues from F4/80 GFP/XMC mice after LPS challenge. The numbers on the X axis of the graph represent numbers assigned to individual F4/80 GFP/Luc transgenic mice in the study.
  • aspects of the invention are useful for understanding microglial cell responses to disease and/or conditions (e.g., injury); identifying molecular pathways associated with microglial cell response to disease or injury; identifying cells and/or molecules that can be targeted to increase or decrease microglial cell response; and for screening, identifying , or evaluating compounds for their effect on microglial cell activity.
  • aspects of the invention can be performed in vivo using transgenic animals or in vitro using cells harboring an inducible reporter for microglial activity.
  • the invention in part, relates to constructs, transgenic cells, transgenic cell lines, and transgenic non-human animals that can be used to monitor macrophage (e.g., microglial) activation.
  • the invention includes, in part, methods of monitoring and assessing microglial activation using a DNA construct with inducible expression.
  • a DNA construct of the invention is also referred to herein as a reporter construct, a genetic construct, a nucleic acid construct, or a reporter transgene.
  • Cells transfected with a genetic reporter construct can be used in assays of macrophage (e.g., microglial) activation.
  • Reporter constructs of the invention can also be used to generate non-human transgenic animals. Constructs may encode a detectable reporter protein.
  • Transgenic cells, and transgenic animals of the invention may include a detectable reporter protein.
  • Constructs, cells and animals of the invention may be designed to express a detectable reporter protein (e.g., GFP or EGFP, or luciferase, etc.) under the direction of a macrophage-specific promoter, (for example, an F4/80 promoter or fragment thereof).
  • a detectable reporter protein e.g., GFP or EGFP, or luciferase, etc.
  • a macrophage-specific promoter for example, an F4/80 promoter or fragment thereof.
  • Constructs, transgenic cells, transgenic cell lines, and transgenic animals of the invention can be used to monitor and assess microglial activation.
  • An F4/80 GWlLuc construct has been designed and used to generate transgenic cells and transgenic mice that expresses enhanced green fluorescent protein (EGFP) and firefly luciferase ⁇ Luc) under the direction of a fragment of the F4/80 promoter.
  • EGFP enhanced green fluorescent protein
  • ⁇ Luc firefly luciferase ⁇ Luc
  • the F4/80 protein is a surface protein expressed exclusively on tissue macrophages, including microglial cells, that is upregulated in response to cellular activation.
  • Transgenic animals of the invention may be used to examine microglial cell biology and the role of microglial cells in neuroinflammatory processes in neurodegenerative disease and disorders.
  • Transgenic mice that carry one or more reporter transgenes of the invention may express the reporter in tissues known to harbor resident macrophages including the brain, spleen, lungs, and liver.
  • reporter protein expression in the brains of the transgenic animals of the invention may be restricted to microglial cells and not detected in astrocytes or neurons.
  • peripheral inflammatory challenge e.g., with 5mg/kg bacterial endotoxin lipopolysaccharide (LPS) BP
  • reporter protein expression robustly and transiently increased in the brain.
  • LPS bacterial endotoxin lipopolysaccharide
  • microglial activation results in luciferase expression in the central nervous system of transgenic animals of the invention after inflammatory challenge or nerve injury.
  • the extent of luciferase activity which can be measured using the Xenogen/IVIS or other suitable imaging system, is proportional to the extent of microglial activation and allows determination of the activation state of microglial cells in the brain or spinal cord in real time.
  • transgenic animals can be used in conjunction with in vivo molecular imaging methods to monitor microglial activation in inflammatory and neuropathological systems. Transgenic animals of the invention are also useful to investigate and/or monitor biomolecular pathways associated with activation of macrophages, including microglial activation.
  • biomolecular pathway may be a receptor- mediated activation of the cell, that is due to engagement of a known ligand for that receptor where receptor-ligand interaction results in triggering of intracellular pathways that alter status of the cell at a transcriptional and/or protein level.
  • Non-limiting examples of such pathways are interleukin 1 acting at the interleukin- receptor, LPS acting at Toll receptor 4, double-stranded RNA acting at Toll receptor 3, adenosine acting at adenosine receptors, including the A2a receptor, and ATP acting at purinergic receptors including P2X4 and P2X7 receptors.
  • Cellular activation may also occur via the interaction of pathogenic proteins associated with neurodegenerative disease and receptors on the macrophage/microglial cell surface.
  • pathogenic proteins include, but are not limited to, amyloid beta, alpha- synuclein and Huntingtin, each of which may activate cells in their native or mutant forms and in different structural states.
  • receptors have been identified that will mediate this response for example amyloid-beta can bind to the scavenger receptor CD36.
  • Transgenic animals of the invention can be used as a source of cells for cell culture.
  • Tissues or cells of transgenic mice can be analyzed for the presence of macrophage activation, either by directly analyzing DNA or RNA, or by assaying the tissue for the reporter protein expressed by the transgene.
  • Cells of tissues carrying the transgene can be cultured using standard tissue culture techniques, and used, e.g., to study the functioning of the transgenic cells.
  • Transgenic animals of the invention may also be used to monitor and examine diseases and/or conditions that are associated with macrophage activation.
  • Some diseases or conditions may be associated with an increase in the activation of macrophage (e.g., microglia) and other diseases or conditions may be associated with a decrease in macrophage (e.g., microglial) activation.
  • diseases and conditions that maybe considered macrophage activation-associated diseases or conditions, include, but are not limited to: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, abnormal development, or aging.
  • the invention in some aspects, also relates to transgenic cells and cell lines in which a detectable expression product is expressed under the control of the F4/80 promoter or a fragment of the F4/80 promoter.
  • Transgenic cells and cell lines of the invention may be derived from a transgenic animal of the invention or may be independently prepared using standard methods known in the art.
  • Cell types that may be suitable and can be used to produce transgenic cells and/or cell lines of the invention can be readily selected by those of ordinary skill in the art, and include, but are not limited to: 239E or 293T cells, BV2 cells, ElAW cells, THP cells.
  • the nucleic acid sequence that encodes a detectable reporter protein may be operably linked to a specific fragment of the F4/80 promoter.
  • the terms “detectable reporter protein” and “detectable expression product” may refer to a product or protein that is directly detectable or indirectly detectable. Indirect detection may include detection based on a reporter protein's activity, as exemplified by the enzymatic activity of luciferase on its luciferin substrate.
  • Transgenic cells and cell lines of the invention are also useful for the study and monitoring of macrophage activation-associated diseases and disorders.
  • the level of expression of a detectable reporter protein in the transgenic cell, cell line, or tissue correlates with the activation of the promoter for F4/80 and macrophage activation in the cell, cell line, or tissue.
  • a transgenic cell, cell line, or tissue prepared with a transgenic construct of the invention can be used to determine the level of expression of F4/80 and macrophage (e.g., microglial) activation.
  • a transgenic cell or cell line, or tissue of the invention can be used as a model for conditions and treatments that modulate or alter macrophage activation and the expression of F4/80 in cells and/or cell lines, and to investigate biomolecular pathways associated with macrophage activation .
  • a specific fragment of a macrophage activation-sensitive promoter region of the F4/80 gene is used to create a macrophage activation reporter, e.g. a DNA reporter construct.
  • the parenchymal microglia are derived from CD45 + bone marrow precursors of myeloid lineage, which give rise to macrophages, dendritic cells, and granulocytes. Parenchymal microglia are macrophage cells that comprise approximately 20% of the non-neuronal population of the central nervous system (CNS). Microglia are extremely sensitive to environmental changes. Microglial activation occurs in a broad spectrum of neurological disorders. Microglial cells are believed to function as the innate immune cells of the brain and spinal cord.
  • microglia form a network throughout the central nervous system (CNS) and are thought to be important in maintaining the CNS in an immune-privileged state.
  • CNS central nervous system
  • ALS amyotrophic lateral sclerosis
  • microglia become activated and may contribute to the pathogenesis of these diseases as well as other neurological diseases and conditions by exacerbating the underlying pathology and in some instances causing neuronal cell death.
  • microglial cells to exacerbate neurodegeneration is thought to be due, at least in part, to their ability to generate and secrete pro-inflammatory molecules such as tumor necrosis factor- ⁇ , interleukin-1 ⁇ , interleukin-6 and nitric oxide (Hanisch, 2002, Gli ⁇ 40:140-155; John et al, 2003, The Neuroscientist 9(1): 10-22).
  • pro-inflammatory molecules such as tumor necrosis factor- ⁇ , interleukin-1 ⁇ , interleukin-6 and nitric oxide (Hanisch, 2002, Gli ⁇ 40:140-155; John et al, 2003, The Neuroscientist 9(1): 10-22).
  • Recombinant nucleic acid constructs that can be used to prepare transgenic cells, cell lines or transgenic animals of the invention may include, at least, a fragment of an F4/80 promoter operably linked to a sequence encoding a reporter (e.g. a reporter protein). Expression of the reporter protein can be driven by the activation of the F4/80 promoter. Surprisingly, it is not necessary to utilize the entire F4/80 promoter region but rather a fragment of the F4/80 promoter can be used in recombinant DNA constructs of the invention.
  • the nucleotide sequence of the promoter is the sequence set forth as SEQ ID NO:2.
  • SEQ ID NO:2 includes nucleotides 1104-2065 of the full-length sequence of the F4/80 promoter having Genbank Accession No. AJ295275 (as posted at the date of filing, and set forth herein as SEQ ID NO: 5). Those of ordinary skill in the art will recognize that variations of the sequence set forth as SEQ ID NO:2 may also be used to generate transgenic cells, cell lines, or animals of the invention. A description of variations of sequence that may be present in a promoter sequence of the invention, such as substitutions, deletions, additions, etc., are provided below herein.
  • two or more different constructs may be used in a transgenic cell or animal of the invention, each expressing a different reporter from an F4/80 promoter or fragment thereof.
  • a functional fragment of an F4/80 promoter is a fragment of the full promoter sequence that, in a construct, cell, cell line, or animal of the invention, that may be operatively linked to a sequence of a reporter protein or fragment thereof, and is effective to result in expression of the reporter protein, or fragment thereof, upon macrophage (e.g. microglial) activation.
  • the term functional fragment may refer to a fragment of a reporter protein, coding sequence for a reporter protein, or fragment thereof.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, or more) sequences that encode a reporter protein that are under the direction of the same promoter may be used in a construct, cell, cell line, or animal of the invention.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, or more) sequences that encode a reporter protein and are under the direction of different promoters can be used in a construct, cell, cell line, or animal of the invention.
  • each construct may be integrated at a different chromosomal location.
  • a fragment of the F4/80 promoter that can be used includes nucleotides 1104-2065 of the full-length F4/80 promoter.
  • fragments of the F4/80 promoter that are of other lengths than SEQ ID NO:2 may also be used in constructs, transgenic cells, transgenic cell lines, and/or transgenic animals of the invention.
  • Fragments of the F4/80 promoter that are longer than SEQ ID NO:2 may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any integer greater than 100, but less than 501 (e.g., 125, 150, 175, 200, 250,
  • a fragment of the F4/80 promoter that is useful in the invention may be shorter than SEQ ID NO:2. Fragments of the F4/80 promoter that are shorter than SEQ ID NO:2, may be shorter by about 1%, 5%, 10%, 15%, 20%, 25%, or more than SEQ ID NO:2. Those of ordinary skill in the art will understand how to prepare additional fragments of the F4/80 promoter for use in constructs, transgenic cells, transgenic cell lines, and/or transgenic animals of the invention using routine procedures.
  • Methods set forth herein for testing the function and ability of reporter construct that includes a fragment of the F4/80 promoter that differs in sequence from SEQ ID NO:2 can be used to compare the activity of the different sequences for use in constructs, transgenic cells, transgenic cell lines, and/or transgenic animals of the invention.
  • the level of macrophage (e.g., microglial) activation in a transgenic cell or animal of the invention in which activity of the macrophage (e.g. microglia) activity has been induced by chemical or injury challenge may be compared to the level of macrophage (e.g.
  • transgenic cells and animals that include the F4/80 fragment that is set forth as SEQ ID NO:2 serves as a control to assess the activity of alternative fragments of the F4/80 promoter in transgenic cells and animals of the invention.
  • a recombinant nucleic acid construct e.g., a plasmid, component of a linearized plasmid that is integrated into genomic DNA, etc.
  • a recombinant nucleic acid construct e.g., a plasmid, component of a linearized plasmid that is integrated into genomic DNA, etc.
  • the regulatory sequences needed for gene expression often include 5'non-coding sequences involved with the initiation of transcription.
  • a coding sequence and regulatory sequences are said to be "operably linked" when they are covalently linked in such a way as to place transcription of the coding sequence under the influence or control of the regulatory sequences.
  • coding sequence and regulatory sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the coding sequence and the regulatory sequences does not (1) result in the introduction of a frame-shift mutation or (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence.
  • a functional promoter region is operably linked to a coding sequence if the promoter region effects transcription of that coding sequence such that the resulting transcript can be translated into the desired protein.
  • the precise nature of the regulatory sequences needed for gene expression in the cells and animals of the invention may vary between species or cell types, but in general may include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Often, such 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • Transgenic animals of the invention may include animals in which one or more cells receive a recombinant DNA molecule.
  • Transgenic animals are genetically modified animals into which cloned nucleic acid material has been experimentally transferred. Cloned genetic material is referred to herein as a transgene.
  • the nucleic acid sequence of the transgene may be integrated at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found. In some embodiments, several copies (e.g., 2, 3,4, or more copies) each may be integrated at a different locus, and may be integrated on different chromosomes
  • Transgenic animals may be homozygous or heterozygous for each integrated transgene.
  • a transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal or cell.
  • Transgenic animals of the invention may include somatic cells that contain a DNA reporter transgene.
  • a transgenic animal may include germ cells that contain the DNA reporter transgene.
  • Methods for generating transgenic cells typically include the steps of (1) assembling a suitable DNA construct useful for inserting a specific DNA sequence into the nuclear genome of a cell; (2) transfecting the DNA construct into the cells; (3) allowing random insertion and/or homologous recombination to occur.
  • the modification resulting from this process may be the insertion of a suitable DNA construct(s) into the target genome; deletion of DNA from me target genome; and/or mutation of the target genome.
  • Genetic constructs e.g., plasmids
  • Constructs also may have suitable origins of replication and/or selectable markers.
  • DNA constructs appropriate for establishing transgenic cells using the materials and methods described herein.
  • Transfection techniques are well known to those of ordinary skill in the art and materials and methods for carrying out transfection of DNA constructs into cells are commercially available.
  • materials that can be used to transfect cells with DNA constructs are lipophilic compounds such as LipofectinTM, activated polycationic dendrimers such as SuperfectTM, LipoTAXITM, and CLONfectinTM. Particular lipophilic compounds can be induced to form liposomes for mediating transfection of the DNA construct into the cells.
  • cationic based transfection agents that are known in the art can be utilized to transfect cells with nucleic acid molecules (e.g., calcium phosphate precipitation).
  • electroporation techniques known in the art can be utilized to translocate nucleic acid molecules into cells.
  • particle bombardment techniques known in the art can be utilized to introduce exogenous DNA into cells. Target sequences from a DNA construct can be inserted into specific regions of the nuclear genome by rational design of the DNA construct.
  • Transgenic animals of the invention can be produced by a variety of different methods including transfection, electroporation, cell gun, liposome fusion, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No.
  • transgenic cells and animals of the invention a gene may be inserted through random insertion but homologous recombination may also be used to generate transgenic cells and animals of the invention.
  • a number of recombinant animals and cells have previously been produced, including, but not limited to: transgenic non-human animals and cells that include a transgene encoding an APP polypeptide comprising the Swedish mutation (U.S. Patent No.
  • mice that express an activated oncogene sequence (U.S. Pat. No. 4,736,866); overexpress aspartyl protease 2 (ASP2) (U.S. Patent No. 6,958,433); express simian SV 40 T-antigen (U.S. Pat. No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRP-I) (U.S. Pat. No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at least one human gene that participates in blood pressure control (U.S. Pat. No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Pat. No. 5,720,936); or have a reduced capacity to mediate cellular adhesion (U.S. Pat. No. 5,602,307).
  • IRP-I interferon regulatory factor 1
  • transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., porcine transgenic animals (U.S. Pat. No. 6,700,037); cloning bovines using reprogrammed non-embryonic bovine cells (U.S. Patent No. 6,011,197); Kim et al., MoI. Reprod. Dev. 46(4):515 526 (1997); Houdebine, Reprod.
  • a commonly used protocol includes direct injection of the transgene into the male pronucleus of the fertilized egg (see Hogan et al., "Manipulating the mouse embryo", Cold Spring Harbor Laboratory, Cold Spring Harbor (1994), resulting in the random integration into one locus of a varying number of copies.
  • the injected eggs are then re-transferred into the uteri of pseudo-pregnant recipient mothers.
  • Some of the resulting offspring may have one, two, three, or more copies of the transgene integrated into their genomes, with integration at one integration site, or at multiple sites throughout the genome.
  • transgenes can be introduced via embryonic stem (ES) cells, using electroporation, retroviral vectors or lipofection for gene transfer. This may be followed by random insertion or insertion by homologous recombination into the genome of the pluripotent embryonic stem (ES) cells, followed by the production of chimeric mice and subsequent germline transmission. Transgenes of up to several hundred kilobases of DNA have been used to produce transgenic mice with this method. This approach can be tailored such that the transgene is inserted into a predetermined locus (non-randomly) that supports ubiquitous as well as tissue-specific expression of the transgene.
  • the methods of introducing a recombinant construct/transgene into mammals and their germ cells were originally developed in the mouse. These methods were subsequently adopted for use with larger animals, including livestock species.
  • Microinjection of DNA into the cytoplasm of a zygote can also be used to produce transgenic animals.
  • Introduction of a recombinant DNA molecule at the fertilized oocyte stage ensures that the introduced gene will be present in all the cells of the transgenic animal. Because the introduced gene will also be present in the germ cells of the transgenic founder animal, all of the founder animal's offspring will carry the introduced gene in all of their cells. Introducing the gene at a later embryonic stage might result in the absence of the introduced gene in some somatic cells of the founder animal, but the offspring of such an animal that inherit the introduced gene will carry the gene in all of their germ cells and somatic cells.
  • non-human animal includes any non-human mammal and other non-human animals.
  • Non-human animals include but are not limited to: non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, rats, etc.
  • a mouse is a preferred mammal species for producing transgenic animals
  • other non-limiting examples of animals that may be used include guinea pigs, rabbits, pigs, sheep, etc.
  • the choice of transgenic animal may be limited by the ability to detect the expression of the reporter protein.
  • a light-generating reporter is utilized and the need for detection of the generated light at the surface of the animal may be used to guide one of ordinary skill in the art to select an appropriate type of animal for use.
  • Non-invasive, high resolution, small animal imaging systems have been developed over recent years employing targeted, activatable molecular imaging reporter gene/reporter probe systems (Massoud & Gambbir, 2003, Genes and Development 17:545-580).
  • the firefly luciferase gene ⁇ Luc encodes firefly luciferase, an enzyme that oxidizes its substrate D- mciferin to result in high light emission (bioluminescence). This chemiluminescent reaction only takes place in living cells.
  • fluorescent reporter proteins e.g., GFP
  • bioluminescent markers do not require an external source of light for excitation.
  • molecular imaging techniques allow monitoring the location, magnitude and persistence of reporter gene expression in intact living animals.
  • a luciferase reporter protein in a transgenic cell, tissue, organ, or animal of the invention, the cell or animal is contacted with, or administered an effective amount of luciferin to allow light to be emitted by oxidation of the luciferin by the expressed luciferase.
  • the amount of light emitted is be proportional to the amount of luciferase expressed, which in turn reflects the amount of macrophage activation in the cell and/or animal of the invention.
  • Luciferase activity is relatively unstable in vivo and reductions in luciferase rnRNA abundance are reflected by reduced luciferase activity over several hours.
  • the glow resulting from luciferase catalysis of its substrate luciferin is widely used in in vitro cell studies as an assay for luc expression which acts as a reporter for the activity of a regulatory element that controls its expression.
  • Luciferase is particularly useful as a reporter because low- light cameras can detect bioluminescence in real time and with high sensitivity in living cells and organisms.
  • a reporter maybe firefly luciferase, such as that isolated from Photinus Pyralis (Luc); Renilla luciferase from sea pansy Renilla reniformis (RLuc); or a luciferase from Japanese Genji-botaru Luciola cruciata (JLuc).
  • the sequence that encodes a luciferase reporter may be a naturally occurring luciferase sequence or may have nucleotide or amino acid sequence that is modified from that of a naturally luciferase sequence.
  • sequence modifications that may be used in a reporter sequence of the invention may include deletions, insertions, etc, some of which are described below herein.
  • luciferase enzyme can incorporate modified versions of the luciferase enzyme, luciferase enzyme from different species or any other protein that can produce light able to cross animal tissues or any enzyme that can emit light able to cross animal tissues when provided with a suitable substrate.
  • the genes encoding such proteins, or variations or such proteins that also retain their detectable function, can be used as a part of a macrophage activation reporter fusion construct of the present invention.
  • variants of a reporter sequence may include sequence modifications and modified reporter sequences will still be useful in the methods of the invention if they retain the detectable features of the parent sequence.
  • Examples of luciferase nucleic acids and reporter proteins, including variants of naturally occurring molecule sequences, and methods for the design and production of alternative sequences that encode a luciferase useful in the methods, transgenic cells and animals of the invention include, but are not limited to, those described in publications such as U.S. published patent application no. 20050272111; U.S. Patent No. 5,330,906; and U.S. Patent No. 5,670,356; etc.
  • Use of luminescent reporter proteins in the constructs, cells, animals and methods of the invention can be optimized based on parameters such as the tissue thickness for detection, emission wavelength etc. Some features and parameters associated with the resolution of luminescence are described in U.S. published patent application no. 20040139487.
  • Such features and parameters influence the ability of the signal from activated macrophages containing the detectable reporter to be detected through the tissue or animal in which they are expressed.
  • Signal attenuation depends on the wavelength of the light emitted and the tissue properties surrounding the emitting cells. In general, blue-green light (400-590 nm) is strongly attenuated while red to near-infrared light (590-800 nm) is less attenuated. Although most types of luciferase have peak emission at blue to yellow-green wavelengths, the emission spectrum is broad enough that there is also significant emission at red wavelengths (>600 nm) that penetrate quite deeply into tissue. For small rodents such as mice, this allows detection of signals throughout the entire animal. Those of ordinary skill in the art will be able to optimize the reporter signal used in the methods, cells, and animals of the invention using routine procedures in imaging methods.
  • bioluminescent reporter The limits of light detection in vivo depend on the type of bioluminescent reporter, the surrounding physiology of the animal and, most importantly, on the source depth.
  • CCD Charged Coupled Device
  • bioluminescent cells in animals can be observed from about 1-cm deep, depending on the number and location of the cells.
  • Biorecinescence imaging allows rapid and noninvasive measurements of macrophage (e.g., microglial) activation. Consecutive images from the same animal permit temporal and spatial information throughout an entire experiment instead of only at the end point. Resolution is less than with MRI or PET, but bioluminescence is better suited for high- throughput imaging because it is simple to operate, images are quick to obtain (less than one minute), several animals can be analyzed at the same time and there is no harm to the animal from the substrate luciferin because it is not toxic and it does not induce an immune reaction. Because animals recover well from gas anesthesia they can be repeatedly subject to luciferin administration thus allowing real-time detection of macrophage (e.g., microglial) activation at more than one time point. Thus, the use of luciferin reporters can allow observation of macrophage (e.g., microglial) activation over time and under various circumstances, such as in induced disease states, before and after injury, or before and after other physiological challenge to the animal.
  • macrophage e.
  • a reporter protein of the invention may also be a non-luminescent reporter protein. Reporter proteins, or the activity of reporter proteins in a construct, cell, cell line, or animal of the invention may be detected by any convenient method available to the user. For example, reporter proteins can be detected using biochemical assays or using fluorescence activated cell sorting (FACS) assays.
  • the detectable reporter protein may comprise, or be coupled to a detectable label.
  • detectable labels are available for use such as those that provide direct detection (e.g., fluorescence, colorimetric, or optical, etc.) or indirect detection (e.g., enzyme-generated luminescence, epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, labeled antibody, etc.).
  • direct detection e.g., fluorescence, colorimetric, or optical, etc.
  • indirect detection e.g., enzyme-generated luminescence, epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, labeled antibody, etc.
  • a variety of methods may be used to detect the label, depending on the nature of the label and other assay components. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for using and detecting labels in DNA reporter constructs, transgenic cells, tissues and animals are well
  • Transgene expression in cells, tissues, or animals can be detected by in vivo, ex vivo, or in vitro imaging of luciferase activity, immunohistochemistry, Western blot of expressed reporter protein, or by any other suitable detection method.
  • the detectable expression product may be a fluorescent, luminescent, colorimetric, or enzymatic expression product, hi some embodiments a reporter protein may be GFP, /3-galactosidase, or chloramphenicol acetyltransferase gene (CAT), or other suitable detectable reporter protein.
  • the invention in some aspects, includes the use of a detectable reporter protein that is enhanced green fluorescent protein (EGFP), firefly luciferase ⁇ Luc), or another detectable expression product.
  • EGFP enhanced green fluorescent protein
  • ⁇ Luc firefly luciferase ⁇ Luc
  • the invention provides non-human transgenic animals expressing a fusion reporter construct that comprises one or more copies of a promoter that is sensitive to the cellular activation of tissue macrophages, and the promoter sequence is operably linked to a reporter gene coding for a detectable protein when there is cellular activation of tissue macrophages.
  • tissue macrophages e.g., activation of microglia
  • the increased expression of the detectable reporter allows the use of the animal to detect modulations in pathways associated with macrophage activation.
  • Transgenic animals of the invention can be challenged with an inflammatory stimulus (e.g., peripheral LPS injection) or a pathological stimulus, (e.g., peripheral nerve injury) and changes in the expression of the detectable reporter protein over time can be assessed, hi some embodiments, expression of the reporter protein can be assessed in brain and spinal cord tissue of the animal and the animal can be used to assess the role and activity of microglial cells in a neurological disease or condition that can be modeled in the animal.
  • an inflammatory stimulus e.g., peripheral LPS injection
  • a pathological stimulus e.g., peripheral nerve injury
  • Transgenic cells or animals of the invention are not more likely to have macrophage activation than their non-transgenic counterparts. Therefore, transgenic cells or animals of the invention can be used to assess the potential of activity or insult to the cell or animal to result in macrophage activation, hi addition to use in detecting changes in macrophage (e.g., microglial) activation, the transgenic cells and animals of the invention also are useful to monitor and assess the ability of a candidate compound to modulate activation of macrophages (e.g., microglia).
  • a candidate compound to modulate activation of macrophages (e.g., microglia).
  • specific models of pathological tissues or cells, disease or injury states, or models of aging, etc. may be generated using cells and/or animals that include a macrophage reporter construct of the invention.
  • Such cells, tissues, cell lines, and/or animals also may include one or more disease phenotypes, such as Alzheimer's disease, Parkinson's disease, etc.
  • a disease state maybe induced in a transgenic cell or animal of the invention by contacting the cell or animal with a disease-inducing or condition-inducing agent or molecule such as an inflammation-inducing agent (e.g., LPS, lipotechoic acid), an apoptotic agent, a toxin, bacteria, virus, heavy metal, etc.
  • a disease-inducing or condition-inducing agent or molecule such as an inflammation-inducing agent (e.g., LPS, lipotechoic acid), an apoptotic agent, a toxin, bacteria, virus, heavy metal, etc.
  • Additional agents that induce inflammatory response in mammals will be known to those of ordinary skill in the art, and can be administered in effective amounts to induce inflammatory response using art-known, routine procedures.
  • agents include inflammatory cytokines such as interleuin-1, tumor necrosis factor-alpha, and inflammatory mediators such as prostanoids, leukotrienes, kinins, purinergic agonists (eg. ATP) and pathogenic proteins (eg Amyloid-beta).
  • inflammatory cytokines such as interleuin-1, tumor necrosis factor-alpha
  • inflammatory mediators such as prostanoids, leukotrienes, kinins, purinergic agonists (eg. ATP) and pathogenic proteins (eg Amyloid-beta).
  • Those of ordinary skill in the art will understand how to administer such agents, and how much to administer to induce an inflammatory response.
  • Constructs, transgenic cells, transgenic cell lines, and transgenic animals of the invention can be used in methods to assess candidate compounds and/or treatments for macrophage activation-associated diseases and/or conditions.
  • candidate pharmaceutical agents or compounds may be tested using transgenic cells or transgenic animals of the invention to determine whether the agent or compound modulates (inhibits or enhances) macrophage activation.
  • a candidate compound can also be tested using transgenic cells, cell lines, and animals of the invention to determine a therapeutically effective amount of the compound, which is that amount effective to modulate macrophage activation.
  • a preferred agent is one that inhibits macrophage activation, rn certain - -
  • a preferred agent is one that enhances macrophage activation. Additional tests useful for monitoring the onset, progression, and/or remission, of a macrophage activation associated with a disease or condition such as those described herein, can be performed using transgenic cells, cell lines, and/or animals of the invention.
  • the desired response is inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease.
  • the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.
  • Transgenic animals of the invention can be crossbred with other animal models of disease or conditions for many purposes, including research.
  • the offspring may be heterozygous or homozygous for the transgene or can be bi-transgenic (carrying two different transgenes) or multi-transgenic [carrying multiple different transgenes, e.g., 3, 4, 5, 6, 7, 8, 9, 210, etc, each independently present in one or more copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. )].
  • disease or condition-prone animals can be generated that possess an intrinsic ability, under the adequate conditions mentioned, to generate reporter protein expression in cells and tissues in which macrophage (e.g., microglial) activation occurs.
  • macrophage e.g., microglial
  • Examples of the types of animals comprising models of diseases or conditions that may be crossed with a transgenic animal of the invention includes, but are not limited to Alzheimer's disease, Parkinson's disease, ALS, and other diseases and conditions described herein and known to those of ordinary skill in the art.
  • Transgenic cells and animals with a macrophage (e.g., microglial) activation reporter sequence of the invention can be used, as a non-limiting example, to: (1) assess one or more biomolecular pathways associated with a neurological disease, condition, or injury in the transgenic cells or animals; (2) determine the effect of candidate compounds on macrophage activation in the cells or animals; (3) study the sequence of events that are associated with macrophage activation including, but not limited to identifying triggers of activation, time- course of activation, etc.; (4) serve as a bioassay system for testing potential macrophage (e.g., microglial) activating compounds; (5) serve as a bioassay system for testing candidate compounds to inhibit or enhance macrophage (e.g., microglia) activation; (6) test other therapeutic compounds to evaluate potential toxicity or side effects on the CNS (e.g. via - -
  • microglial activation or inhibition examples of other therapeutic compounds may include, but are not limited to, therapeutic compounds designed to prevent or treat diseases such as cancer, cardiovascular disease, neurological disease, injury, etc.
  • Transgenic cells and animals of the invention provide in vivo models for testing methods and compounds for inhibiting or enhancing macrophage activation and for testing novel therapeutic modalities including pharmaceutical compounds, surgical treatments, gene therapy, immunotherapy etc.
  • the uses mentioned above can also be derived from transgenic cell or animals that do not possess the ability to report macrophage activation through light emission, the added ability of bioluminescence greatly facilitates such uses of the macrophage-activation models. Fewer animal subjects are required to obtain statistically meaningful results and because the data obtained reveal functional information, animal studies can be refined. Moreover, because the studies can be performed in minimal disease states, the stress on the animals that are studied can be reduced.
  • a transgenic animal in which a detectable expression product e.g., EGFP or firefly luciferase, etc
  • a detectable expression product e.g., EGFP or firefly luciferase, etc
  • F4/80 promoter or a fragment of an F4/80 promoter can be used as a model system to monitor the activation state of microglia in real time.
  • Transgenic animals, cells, and tissues of the invention can be used as models of neurodegenerative diseases and conditions and to determine the level of microglial activation (e.g., neuroinflammation) in the animal, cell, or tissue under various disease or normal conditions.
  • Such diseases or conditions may include, but are not limited to: Alzheimer's disease, Parkinson's disease, Huntington's disease, infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, abnormal development, or aging.
  • a transgenic animal may include at least one inducible reporter gene and at least one genetic modification that results in a model disease state in the animal.
  • a transgenic animal comprising at least one inducible reporter gene may be crossed with an animal carrying at least one genetic modification that results in a model disease state in the animal.
  • the disease is a disease such as ALS, Alzheimer's disease, etc, or other disease described herein.
  • such an assay may include providing a transgenic animal that includes a reporter construct with 1) at least one reporter sequence that encodes a detectable reporter protein, and 2) at least one promoter that drives the expression of the detectable reporter. Promoter-driven expression of the reporter protein of the construct is induced by macrophage activation.
  • the transgenic animal may have a macrophage-associated disease or disorder, and/or may be treated with a macrophage-inducing compound or treatment that results in abnormal macrophage activation.
  • Such an assay may also include detecting expression of the reporter.
  • the reporter may be a luciferase protein and the animal is administered luciferin, the substrate for luciferase, in an amount effective to detect the presence and/or level of the expression of the reporter protein.
  • a reporter protein may be another detectable protein.
  • the level of macrophage activation (for example neuroinflammation) in a normal transgenic animal of the invention maybe compared to the level of macrophage activation in a transgenic animal of the invention that has a neurological disease or condition.
  • the term "normal” means "disease or condition free.”
  • the level of macrophage activation in an transgenic animal that is free of Alzheimer's disease may be compared to the level of macrophage activation in a transgenic animal that has an Alzheimer's disease-associated genotype or phenotype.
  • a transgenic animal of the invention can be used to determine or monitor the effect of a candidate therapeutic agent or other treatment regimen (e.g., surgery, heat treatment, etc) on the level of macrophage activation in a cell, tissue, or animal.
  • a candidate therapeutic agent or other treatment regimen e.g., surgery, heat treatment, etc
  • the level of expression of the detectable reporter protein e.g., EGFP, GFP, or luciferase, etc
  • the detectable reporter protein e.g., EGFP, GFP, or luciferase, etc
  • the level of expression of the detectable reporter protein correlates to the level of macrophage activation (e.g.
  • a reduction in the expression of the detectable reporter protein may mean the reduction in the expression of F4/80 and reduction in macrophage activation and conversely, an increase in the expression of the detectable reporter protein may mean an increase in the expression of F4/80 and increase in macrophage activation.
  • a transgenic animal which comprises a transgene comprising a promoter of a macrophage-specific protein directing the expression of one or more reporter proteins.
  • the transgenic animal is - -
  • the macrophage-specific promoter comprises the F4/80 fragment provided herein as SEQ ID N0:2 or a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the F4/80 fragment provided herein as SEQ ID NO:2.
  • the reporter protein of the invention is GFP, EGFP, or luciferase.
  • a transgenic mouse of the invention comprises the F4/80 fragment provided as SEQ ID NO:2 operatively linked to a luciferase reporter coding sequence
  • a transgenic mouse of the invention comprises the F4/80 fragment provided as SEQ ID NO:2 operatively linked to a luciferase reporter coding sequence and to an EGFP reporter coding sequence.
  • the methods, constructs, transgenic cells, transgenic cell lines, and transgenic animals of the invention can be used to determine the level of macrophage activation in a cell, tissue, or animal and may be used to diagnose macrophage activation-associated diseases or conditions.
  • the methods of the invention involve determining the level of reporter protein expression in a transgenic cell, tissue, or animal of the invention as a measure of the level of macrophage activation in the cell, tissue, or animal.
  • the methods are therefore useful to detect a difference in the level of macrophage activation in a transgenic cell, tissue, or animal compared to a control level of macrophage activation.
  • macrophage activation-associated diseases or conditions means a condition or disease in which macrophage activation is either abnormally high or abnormally low.
  • Embryonic and post- embryonic development and tissue repair processes are examples of a macrophage activation- associated condition because events in each of these types of development maybe associated with either a higher or lower than normal level of macrophage activation in cells and tissues.
  • cellular and molecular pathway events that occur in development and cell repair processes may be associated with alterations in macrophage activation. It will be clear to those of ordinary skill in the art, that not all macrophage activation-associated conditions are abnormal or are indicative of illness.
  • a macrophage activity-associated disease or condition may be an illness, injury, or other abnormal indication in a cell, tissue, or animal. In each case, the disease or condition is associated with macrophage activation, either abnormally high amounts or abnormally low amounts.
  • diseases and conditions that may be considered macrophage activation-associated diseases or conditions, include, but are not limited to: Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), infection, inflammation, stroke, brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, epilepsy, peripheral neuropathy, peripheral nerve injury, a psychiatric disease, normal development, aging, normal cell and tissue development, normal cell and tissue aging, effects of toxin exposure, CNS diseases, metabolic disorders, infections, cell and tissue repair, cancer. In each disease and condition, an alteration in macrophage activation is associated with the state of the cell or tissue.
  • ALS amyotrophic lateral sclerosis
  • Transgenic animals of the invention may also be used to assess therapeutic strategies including, but not limited to: dosage requirements; timing of administration; delivery methods and molecules; targeting strategies for delivery to specific cell, tissue types, and/or to specific body regions (e.g., across the blood brain barrier) etc.
  • therapeutic strategies including, but not limited to: dosage requirements; timing of administration; delivery methods and molecules; targeting strategies for delivery to specific cell, tissue types, and/or to specific body regions (e.g., across the blood brain barrier) etc.
  • the level of expression of the detectable expressed product may be monitored and compared to a control (e.g., untreated level).
  • a change between the treated and untreated levels may indicate whether the compound reached a target region in the brain, whether an effective amount of the compound was administered, and/or whether a delivery molecule that was administered in conjunction with the compound delivered the compound to the intended target region or tissue, etc.
  • assays for evaluating the ability of a compound to modulate (increase or inhibit) macrophage (e.g. microglia) activation are provided.
  • an assay may include providing a transgenic animal that includes a reporter construct with 1) at least one reporter sequence that encodes a detectable reporter protein, and 2) at least one promoter that drives the expression of the detectable reporter. Promoter-driven expression of the reporter protein of the construct is induced by macrophage activation.
  • the transgenic animal may have a macrophage-associated disease or disorder, and/or may be treated with a macrophage-inducing compound or treatment that results in abnormal macrophage activation.
  • Such an assay may also include detecting expression of the reporter protein.
  • the reporter protein may be a luciferase protein and the animal is administered luciferin, the substrate for luciferase, in an amount effective to detect the presence and/or level of the expression of the reporter protein.
  • a reporter protein may be another detectable protein.
  • Candidate compounds useful in accordance with the invention encompass numerous chemical classes, although typically they are organic compounds.
  • the candidate compounds are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500.
  • Candidate compounds comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules.
  • the candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.
  • Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.
  • the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules are also contemplated.
  • Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological compounds may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the compounds.
  • Levels of macrophage (e.g., microglial) activation in a transgenic cell or animal of the invention can be determined using methods described herein to assess the level of a reporter protein and are advantageously compared to controls according to the invention.
  • a control may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as in groups having normal amounts of macrophage (e.g., microglial) activation and groups having abnormal amounts of macrophage (e.g., microglial) activation. Another example of comparative groups would be groups of transgenic cells or transgenic animals having a particular disease, condition or symptoms and groups without the disease, condition or symptoms.
  • Another comparative group would be a group of transgenic cells or animals that have a family history of a condition and a group without such a family history.
  • the predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups.
  • examples of such groups may include: a low-risk group, amedium- risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being cells or animals with the lowest risk or amount of macrophage activation (e.g., microglial activation) and the highest quadrant or quintile being cells or animals with the highest risk or amounts of macrophage activation (e.g., microglial activation).
  • Ih macrophage-associated disorders and/or conditions that may be characterized by a decrease in macrophage activation compared to normal levels of macrophage activation
  • examples of such groups may include: a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being cells or animals with the lowest risk and highest amount of macrophage activation (e.g., microglial activation) and the highest quadrant or quintile being cells or animals with the highest risk and lowest amounts of macrophage activation (e.g., microglial activation).
  • the predetermined value will depend upon the particular population selected. For example, an apparently healthy population of transgenic cells or transgenic animals will have a different 'normal' range than will a population of transgenic cells or animals that is known to have a condition associated with abnormal macrophage activation. Accordingly, the predetermined value selected may take into account the category in which an individual cell or animal falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
  • "abnormal" means not normal as compared to a control. By abnormally high it is meant high relative to a selected control. By abnormally low it is meant low relative to a selected control. Typically the control will be based on apparently healthy normal cells or animals in an appropriate age bracket. Healthy normal cells or animals may be transgenic normal cells or animals.
  • measures of macrophage activation can be made over time in a single transgenic animal of the invention.
  • a determination of the level of macrophage activation (e.g., microglial activation) in a transgenic animal of the invention may serve as a control for a determination of the level in that transgenic animal taken at a different time.
  • a baseline level of macrophage (e.g., microglial) activation may be obtained for a transgenic animal of the invention and used as a control level to which other determinations of the level of macrophage (e.g., microglial) activity can be compared.
  • the animal may receive an inflammatory challenge, or a candidate treatment, etc.
  • the invention also includes in part, nucleic acid sequences and polypeptide sequences they encode that are useful in the transgenic cells, cell lines and animals of the invention.
  • nucleic acid sequences that are useful in the invention methods, cells, cell lines, and animals are presented herein as the full reporter construct set forth as SEQ ID NO:1 and an F4/80 promoter fragment set used in a construct of the invention as set forth herein as SEQ ID NO:2.
  • the nucleic acid of SEQ ID NO:2 may be used as a promoter that is responsive to microglial activation or inhibition.
  • the nucleic acid of SEQ ID NO:2 may be operably linked to any suitable reporter gene (e.g., including, but not limited to, one or more of those described herein), hi some embodiments, the promoter fragment may be a restriction fragment of a naturally occurring F4/80 promoter comprising a regulatory region corresponding to SEQ ID NO.2, or modified sequence thereof. Modifications of the nucleic acid sequences of the constructs presented herein may be used in constructs, transgenic cells, transgenic cell lines, and/or transgenic animals of the invention.
  • any suitable reporter gene e.g., including, but not limited to, one or more of those described herein
  • the promoter fragment may be a restriction fragment of a naturally occurring F4/80 promoter comprising a regulatory region corresponding to SEQ ID NO.2, or modified sequence thereof. Modifications of the nucleic acid sequences of the constructs presented herein may be used in constructs, transgenic cells, transgenic cell lines, and/or transgenic animals of the invention.
  • the invention in some aspects, includes a construct useful to make a transgenic animal or cell or cell line of the invention as well as sequences such as a fragment of the F4/80 promoter that are useful in the constructs of the invention, and homologs and alleles of any of the sequences provided or referenced herein.
  • homologs and alleles typically will share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide identity and/or at least 95% amino acid identity to the sequences of a all or part of a construct nucleic acid sequence (e.g., promoter nucleic acid sequence, reporter nucleic acid sequence, and/or additional nucleic acid sequences of the construct) and polypeptides, respectively, in some instances will share at least 95% nucleotide identity and/or at least 97% amino acid identity, in other instances will share at least 97% nucleotide identity and/or at least 98% amino acid identity, in other instances will share at least 99% nucleotide identity and/or at least 99% amino acid identity, and in other instances will share at least 99.5% nucleotide identity and/or at least 99.5% amino acid identity.
  • nucleic acid sequence e.g., promoter nucleic acid sequence, reporter
  • the homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Maryland) that can be obtained through the internet.
  • Exemplary tools include the BLAST system available from the website of the National Center for Biotechnology Information (NCBI) at the National Institutes of Health. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the Mac Vector sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
  • PCR polymerase chain reaction
  • PCR primers are selected to amplify portions of a nucleic acid sequence believed to be conserved.
  • nucleic acids are preferably amplified from a tissue-specific library.
  • the invention also includes degenerate nucleic acids that include alternative codons to those present in the native materials and materials of the invention.
  • serine residues are encoded by the codons TCA, AGT, TCC, TCG 5 TCT and AGC.
  • Each of the six codons is equivalent for the purposes of encoding a serine residue.
  • any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating polypeptide.
  • nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG, and CCT
  • amino acid residues may be encoded similarly by multiple nucleotide sequences.
  • the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • the invention also provides modified nucleic acid molecules, which include additions, substitutions and deletions of one or more nucleotides (preferably 1-20 nucleotides).
  • these modified nucleic acid molecules and/or the polypeptides they encode retain at least one activity or function of the unmodified nucleic acid molecule and/or the polypeptides, such as activity as a promoter or reporter sequence, etc.
  • the modified nucleic acid molecules encode modified polypeptides, preferably polypeptides having conservative amino acid substitutions as are described elsewhere herein.
  • the modified nucleic acid molecules are structurally related to the unmodified nucleic acid molecules and in preferred embodiments are sufficiently structurally related to the unmodified nucleic acid molecules so that the modified and unmodified nucleic acid molecules hybridize under stringent conditions known to one of ordinary skill in the art.
  • modified nucleic acid molecules that encode polypeptides having single amino acid changes can be prepared.
  • Each of these nucleic acid molecules can have one, two or three, four, five, six, seven, eight, nine, ten, or more nucleotide substitutions exclusive of nucleotide changes corresponding to the degeneracy of the genetic code as described herein.
  • modified nucleic acid molecules that encode polypeptides having two amino acid changes can be prepared which have, e.g., 2-6 nucleotide changes.
  • Numerous modified nucleic acid molecules like these will be readily envisioned by one of ordinary skill in the art, including for example, substitutions of nucleotides in codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and so on.
  • each combination of two amino acids is included in the set of modified nucleic acid molecules, as well as all nucleotide substitutions which code for the amino acid substitutions.
  • Additional nucleic acid molecules that encode polypeptides having additional substitutions (i.e., 3 or more), additions or deletions (e.g., by introduction of a stop codon or a splice site(s)) also can be prepared and are embraced by the invention as readily envisioned by one of ordinary skill in the art. Any of the foregoing nucleic acids or polypeptides can be tested by routine experimentation for retention of activity or structural relation to the nucleic acids and/or polypeptides disclosed herein.
  • deletion means deletion, addition, and substitution changes to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleic acids of a sequence of the invention.
  • substitution means deletion, addition, and substitution changes to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleic acids of a sequence of the invention.
  • Methods set forth herein for testing the function and ability of modified sequences such as modified promoter sequences, modified reporter sequences and/or modified sequences of any other part of a reporter construct of the invention can be used to compare the activity of the different sequences for use in constructs, transgenic cells, transgenic cell lines, and/or transgenic animals of the invention.
  • the level of macrophage (e.g., microglial) activation in a transgenic cell or animal of the invention in which activity of the macrophage (e.g., microglia) has been induced by chemical or injury challenge maybe compared to the level of macrophage (e.g., microglial) activation in a similarly treated transgenic cell or animal that includes a modified sequence, e.g.
  • transgenic cells and animals that include the construct set forth as SEQ ID NO: 1 may serve as a control to assess the activity of reporter constructs with modified sequences in transgenic cells and animals of the invention.
  • a kit of the invention may include a reporter construct, transgenic cell, transgenic cell line, or transgenic animal of the invention and instructions for use.
  • the kits can further contain at least one additional reagent, such as luciferin, one or more antibodies or other reporter protein detection means.
  • Kits containing a construct, transgenic cell, transgenic cell line, or transgenic animal of the invention can be prepared for in vivo, ex vivo, and/or, in vitro diagnosis, prognosis and/or monitoring a macrophage activation-associated disease or condition using the optical detection methods described herein or using another suitable immunohistological or immunocytological methods known in the art.
  • the components of the kits may be packaged frozen, chilled, or at room temperature, and certain components may be packaged in aqueous medium or in lyophilized form.
  • a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, cages, or the like.
  • a first of said container means or series of container means may contain one or more constructs, transgenic cells, or transgenic animals of the invention.
  • a second container means or series of container means may contain luciferin or other means of detecting expression of a reporter protein that is expressed in the cell, cell line, or animal with macrophage activation.
  • a kit of the invention may also include a control.
  • the kit also may further comprise instructions as described above.
  • the instructions typically will be in written form and will provide guidance for carrying out the assays embodied by the kit and for making a determination based upon that assay.
  • Example 1 F4/80GFP/Luc construct generated for transgenesis and the generation of F4/80 GFP/Luc transgenic mice.
  • a nucleic acid construct was prepared that includes a fragment of an F4/80 promoter that is operably linked to a reporter sequence.
  • the full-length F4/80 promoter sequence has Genbank Accession No. AJ295275.
  • the fragment of the F4/80 promoter used in the constructs and transgenic animals and cells included nucleotides 1104-2065 of the sequence of Accession No. AJ295275.
  • the sequence of the F4/80 promoter fragment is provided herein as SEQ ID NO:2.
  • the F4/80GFP/Lwc construct is illustrated in Fig. 1 and the nucleotide sequence of the full reporter construct is set forth as (SEQ ID NO: 1).
  • Transgenic mice were generated on a C57B1/6 background by conventional techniques. Briefly, transgenes were injected into pronuclei of C57B1/6 fertilized eggs. Transgenic mice generated were maintained under specif ⁇ c-pathogen-free (SPF) conditions. Integration of the transgene was determined by polymerase chain reaction (PCR) analysis of tail biopsy DNA, amplifying the EGFP gene using primers 5'-caagggcgaggagctgttca (forward; SEQ TD NO:3) and 5'-gatgccgttctctgctgtgtgt (reverse; SEQ ID NO:4). Founder mice were crossed to C57B1/6 mice to produce Fl transgenic and wild-type littermates. Offspring were characterized by in vivo imaging of luciferase activity, ex vivo analysis by immunohistochemistry, Western Blot and FACS, and inducibility of transgene expression determined following inflammatory and pathological stimuli.
  • SPF pathogen-free
  • Transgenic mice expressing the F4/80 EGFP/Luc construct could be differentiated from their wild type littermates by imaging. Five to six-week-old mice were injected with luciferin (150mg/kg ip) and 10 min later imaged using the Xenogen/TVlS imaging system. Baseline expression of luciferase could clearly be determined in transgenic offspring compared to their wild type littermate controls.
  • Example 2 Ex vivo tissue distribution of the F4/80 GFP/Luc transgene by imaging.
  • mice necropsied lOmin after luciferin injection. Organs were rapidly removed and placed in a luciferin bath and imaged less than 20min post mortem. Luciferase expression was observed in brain, spinal cord, lung, and testes. In addition, GFP expression was determined in microglial cells in the brains of F4/80-
  • EGFP/Luc reporter mice Brains of founder mice were removed and processed for histochemical analysis by fixation in 4% paraformaldehyde/10% neutral buffered formalin (4%PFA/10%NBF), incubation in 30% sucrose and cryosectioned at a thickness of lO ⁇ m. Brain sections were stained with markers for neurons (NeuN), Astrocytes (GFAP) or microglia (F4/80 or ⁇ 4 integrin). Only cells expressing GFP stained positively with immunohistological microglial markers. Neurons and astrocytes did not express GFP.
  • Neurons and astrocytes did not express GFP.
  • GFP expression was also determined in tissue macrophages in peripheral organs.
  • Heart, lungs, liver, spleen, and skin samples were viewed by fluorescent microscopy to determine GFP expression.
  • Tissues were also stained with markers of tissue macrophages to identify cells expressing the transgene in peripheral organs. GFP-positive cells were observed in lungs, liver, and spleen.
  • LPS Peripheral injection LPS (5mg/kg ip) resulted in increased expression of luciferase.
  • Five to six-week-old transgenic mice and wild type littermate controls were shaved over the head and dorsal surface of the spinal cord and belly then imaged for baseline luciferase activity from a dorsal and ventral perspective.
  • LPS (5mg/kg) was injected into the peritoneum and the mice were imaged 4h, 6h, 8h, 24h, 72h, and 5 days after LPS treatment. In each instance luciferin was injected (150mg/kg IP) 10 min prior to imaging.
  • Peripheral nerve injury resulted in increased expression of luciferase in the dorsal horn of the spinal cord
  • mice were imaged for baseline luciferase activity and then underwent sciatic nerve crush injury. Mice were then imaged 1, 3, 6, 8, 10, 13, and 15 days after injury following peripheral injection of luciferin (150mg/kg) and luciferase activity was shown to increased in the dorsal horn of the spinal cord in a time-dependent manner (Fig. 3).
  • mice were treated with the tetracycline derivative minocycline (50mg/kg i.p) that has been reported to inhibit microglial activation.
  • Mice were injected with rninocyclme(-24h and daily thereafter) then challenged with LPS (5mg/kg IP) and imaged 4, 8, 12, 24, 48, and 72 h later after injection of luciferin (150mg/kg IP).
  • LPS 5mg/kg IP
  • luciferin 150mg/kg IP
  • Treatment with minocycline attenuated the induction of luciferase by LPS (Fig. 4).
  • F4/80 GFP/Z «c transgenic mice were injected with
  • mice were challenged with LPS (5mg/kg EP) or PBS and 24h later tissues harvested and homogenized to assay for luciferase activity using a commercially available kit (Promega,
  • a reporter mouse was generated by specifically labeling a tissue macrophage-specific promoter (F4/80) that is only expressed in tissue macrophages, namely liver, kidney, spleen, skin epidermis, thymus and brain (McKnight & Gordon, 1996, Immunol. Today 17:283-287).
  • F4/80 (EGF-TM7) is a cell surface glycoprotein with no known function (McKnight & Gordon, 1996, Immunol. Today 17:283-287) because mice lacking the F4/80 gene have no discernible phenotype (Schaller et al, 2002, MoI. Cell Biol. 22(22):8035-43).
  • the reporter construct that was generated contained the sequences for enhanced green fluorescent protein (EGFP) and luciferase in addition to a 960 base pair region of the F4/80 promoter sequence (Fig. 1).
  • luciferase Baseline expression of luciferase was detected in mice carrying the reporter transgene after peripheral injection of luciferin using the IVIS Xenogen imaging system. Expression of the transgene was limited to tissues known to contain tissue macrophages expressing F4/80. Upon activation microglial cells increase their expression of EGFP and luciferase. EGFP expression allowed the identification of microglial cells ex vivo by histological analysis of brain sections under a fluorescent microscope or isolation of microglial cells by FACS. Luciferase expression could be monitored in vivo in real time after peripheral injection of luciferin using the IVIS Xenogen imaging system.
  • mice were challenged with an inflammatory stimulus, namely peripheral LPS injection (5mg/kg i.p), or a pathological stimulus, namely peripheral nerve injury, and changes in luciferase expression over time in the brain and spinal cord were recorded.
  • an inflammatory stimulus namely peripheral LPS injection (5mg/kg i.p)
  • a pathological stimulus namely peripheral nerve injury
  • mice The invention assessment of the role of microglial cells over the course of any neurological disease that can be modeled in mice. Specifically, these reporter mice allow tracking in real time of the onset of microglial activation in vivo and also allows the rapid isolation of activated cells from the CNS for ex vivo analysis (for example transcriptional profiling or proteomics).
  • mice described herein are bred to other genetically modified mice to study the role of microglial cell activation in neurodegenerative disease.
  • F4/80 GFP/Luc mice are crossed with 3XTg-AD mice harboring PSl M i4 6 v 5 APP Sw e and Tau P3 oiL mutations (Oddo et al, 2003 Neuron 39:409-421), an inducible transgenic mouse overexpressing p25 (Cruz et al, 2003 Neuron 40:471-483), a CDl Ib-HSVTK transgenic mouse expressing herpes simplex thymidine kinase in macrophages and microglia (Heppner et al, 2005 Nat. Med.
  • mice expressing a dominant negative form of IKB off the GFAP promoter (Brambilla et al, 2005 J. Exp. Med. 202:145-156.) or mice expressing mutant forms of human SODl (eg. SODl G93A) (Gurney et al, 1994 Science 264:1772-5).
  • the mice described herein are used to screen for pharmacological agents that inhibit microglial activation (for example anti-inflammatory agents) and/or to identify novel biological pathways involved in disease pathology that may provide tractable approaches to the treatment of a wide variety of chronic and acute neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis (ALS), stroke, etc.
  • pharmacological agents that inhibit microglial activation for example anti-inflammatory agents
  • ALS Amyotrophic lateral sclerosis

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Abstract

Dans certains aspects, l'invention concerne des constructions d'acides nucléiques, des cellules transgéniques, des animaux transgéniques non humains contenant une protéine rapporteuse dont l'expression est induite par l'activation des macrophages. L'invention concerne également en partie l'utilisation de ces constructions d'acides nucléiques, cellules transgéniques, animaux transgéniques non humains dans la définition du statut d'activation des macrophages dans des cellules, tissus et animaux présentant une activation des macrophages induite ou une maladie ou un trouble lié(e) à l'activation des macrophages. Dans certains aspects, l'invention concerne encore des méthodes d'identification de composés renforçant ou inhibant l'activation des macrophages, et d'identification et de contrôle de voies biomoléculaires associées à l'activation des macrophages.
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