WO2014045674A1 - Adn possédant une activité promotrice dépendante de l'activité nerveuse, et vecteur contenant celui-ci - Google Patents

Adn possédant une activité promotrice dépendante de l'activité nerveuse, et vecteur contenant celui-ci Download PDF

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WO2014045674A1
WO2014045674A1 PCT/JP2013/068289 JP2013068289W WO2014045674A1 WO 2014045674 A1 WO2014045674 A1 WO 2014045674A1 JP 2013068289 W JP2013068289 W JP 2013068289W WO 2014045674 A1 WO2014045674 A1 WO 2014045674A1
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activity
dna
cell
sare
expression
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晴彦 尾藤
浩行 奥野
尚之 川島
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国立大学法人東京大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • 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)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to DNA that generates promoter activity depending on nerve activity, a vector containing the same, and the like.
  • Cognitive activity which is the essence of higher brain functions, is based on neural activity in the brain. For this reason, attempts to identify the site of neural activity associated with specific cognitive activity have been made by electrophysiological techniques and functional nuclear magnetic resonance imaging, but the former lacks spatial information on signals, while the latter Neural activity mapping at the cellular level is impossible because changes in blood flow are taken as signals.
  • neural activity mapping has also been attempted using the expression of various immediate-early genes (IEG) such as c-fos or Arc / Arg3.1, and modification of transcription factors such as CREB phosphorylation.
  • IEG immediate-early genes
  • modification of transcription factors such as CREB phosphorylation.
  • An attempt is made to express the reporter protein in a stimulus-responsive manner using the IEG promoter derived from the c-fos gene or Arc / Arg3.1 gene, and imaging using the IEG promoter labels a highly active site in the brain It was suggested that they could be used as non-patent documents 2 and 5-7.
  • the present inventors analyzed in detail the expression control region of Arc / Arg3.1 whose expression level changes with neural activity of the brain, designed a promoter in which several fragments thereof were linked in tandem, and downstream A viral vector containing an expression cassette linked to a reporter gene was prepared. And when this virus vector was infected with nerve cells, the reporter gene was driven by a promoter and expressed in response to cognitive activity with a sensitivity 10 times higher than the previous report, and it was possible to visualize neural activity in the cortical region. confirmed.
  • the present invention [1] DNA having a neural activity-dependent promoter activity, Among the synaptic activity responsive elements (SARE) on the promoter of Arc / Arg3.1 gene, a fragment containing CREB binding site, MEF2 binding site, and SRF binding site contains a construct linked at least twice in tandem. DNA; [2] The neural activity-dependent promoter activity according to [1] above, wherein the fragment containing the CREB binding site, MEF2 binding site, and SRF binding site among the SAREs comprises a construct linked 5 times in tandem.
  • SARE synaptic activity responsive elements
  • DNA having neural activity-dependent promoter activity DNA in which any of the following (i) to (iv) includes a construct linked in tandem at least twice: (i) DNA consisting of the base sequence represented by SEQ ID NO: 2, 3, or 4; (ii) DNA consisting of a sequence in which one or more bases are deleted, added or substituted in the base sequence represented by SEQ ID NO: 2, 3, or 4; (iii) DNA comprising a sequence having 70% or more identity with the base sequence represented by SEQ ID NO: 2, 3, or 4; (iv) DNA capable of hybridizing under stringent conditions with a nucleic acid having a sequence complementary to the base sequence represented by SEQ ID NO: 2, 3, or 4; [4] The DNA having a neural activity-dependent promoter activity according to claim 3, wherein the DNA of any one of (i) to (iv) comprises a construct linked in 5 tandems; [5] The DNA having a nerve activity-dependent promoter activity according to any one of [1] to [4] above, wherein the
  • a reporter assay When a reporter assay is performed using the promoter according to the present invention, it is possible to visualize nerve cells that are activated in accordance with cognitive activity, and to easily map the site of nerve activity. In addition, if a reporter assay is performed using the promoter according to the present invention, it is possible to know the presence / absence and strength of activity of a specific nerve cell. Therefore, it can also be used as a marker for confirming whether stem cells have differentiated into nerve cells, a marker for indicating the survival or death of nerve cells, and a marker for cell-level neuroplasticity.
  • FIG. 1A shows the genomic components used in the construction of E-SARE.
  • the top is a map of the Arc / Arg3.1 promoter.
  • SARE is indicated by the leftmost box
  • ArcMin minimum promoter including TATA
  • the other box shows an evolutionarily conserved genomic domain.
  • the bottom row is the sequence of SARE elements from three different species (mouse, human, bovine). Evolutionarily conserved sequences are shown in gray.
  • Each transcription factor binding site is indicated by the name of the transcription factor.
  • the fragment used to construct the E-SARE is indicated by three double arrows.
  • FIG. 1B shows the design and operation of E-SARE. The promoter design is shown in the upper part.
  • FIG. 1A Single or multiple fragments of SARE shown in FIG. 1A were linked to ArcMin, and the transcriptional activity in cultured neurons was measured by luciferase assay.
  • the lower panel shows the transcriptional activity of each promoter when cells are silent (TTX, blue) and when stimulated (4AP & BIC, red).
  • the one with the highest expression level was named E-SARE.
  • FIG. 1C shows a comparison of the capabilities of E-SARE and other neural activity dependent artificial promoters (using multiple multiplexed individual elements within the promoter module).
  • 5xCRE is repeated 5 times for the CREB binding site.
  • 5xMRE is repeated 5 times for the MEF2 binding site.
  • FIG. 1E is a representative image of d2EGFP fluorescence driven by E-SARE or c-Fos promoter in cultured neurons.
  • FIG. 2A shows a simplified transcription model by E-SARE consisting of a short initial phase and a long late phase.
  • the early phase shows a pulse-like strong expression (a1) for a short time (t1), and the late phase shows a weakly decaying weak expression (a2) over a long time (t2).
  • FIG. 2B shows the experimental schedule used to measure transcriptional kinetic parameters with E-SARE and Arc promoters.
  • FIG. 2C shows the results of the luciferase assay at multiple time points.
  • 1xFrgA indicates 1xFragment A (see FIG. 1A).
  • Arc7000 shows the full length of a 7-kb promoter spanning from ArcMin to SARE (see FIG. 1A).
  • FIG. 2D shows the result of normalizing the data of FIG. 2C. Each trace was obtained by dividing the time course of each promoter by the average of each trace. Multiple comparisons were made using values 4 or 24 hours after stimulation. The results are shown as (significant difference after 4 hours, significant difference after 24 hours). ** P ⁇ 0.01. ns is not significantly different.
  • FIG. 2E shows the parameter redundancy of the test model using the maximum likelihood method. Each panel shows a two-dimensional intercept of the parameter space plotting the likelihood of the two parameter combinations described above with the other parameters fixed at the optimum values. A black dot indicates an optimum value.
  • FIG. 2F shows a comparison of transcriptional kinetic parameters obtained in silico and in situ. The dynamics of simulation transcription, mRNA expression and protein expression overlapped well with those obtained from the measurement data.
  • FIG. 3A shows the design of the E-SARE double cassette AAV vector. ITR is inverted terminal repeats; d2Venus is destabilized YFP (Venus); pA is polyadenylation signal; cHS4 is chick insulator HS4; E-PGK is an enhanced PGK promoter; WPRE is after transcription of woodchuck hepatitis virus Each regulator is shown.
  • ITR is inverted terminal repeats
  • d2Venus is destabilized YFP (Venus)
  • pA is polyadenylation signal
  • cHS4 is chick insulator HS4
  • E-PGK is an enhanced PGK promoter
  • WPRE is after transcription of woodchuck hepatitis virus Each regulator is shown.
  • FIG. 3B shows the method of bilateral injection of E-SARE AAV into the mouse primary visual cortex. Only one hemisphere was activated by monocular visual stimulation. Contra indicates the opposite side; Ipsi indicates the same side.
  • FIG. 3D shows an example in which RFP fluorescence is detected from above the skin in an E-SARE AAV-infected cortical region.
  • FIG. 3E shows an observation example with the naked eye from the back of the brain of an animal that received E-SARE AAV.
  • FIG. 3F is a fluorescence image of a coronal section of the brain for histological confirmation of neural activity-dependent reporter expression.
  • the Roman numerals on the left indicate the cortical layer.
  • the fluorescence signals of RFP and GFP are shown as native fluorescence signals without immunohistological enhancement.
  • the scale bar is 100 ⁇ m.
  • FIG. 4A shows 3D stacking of images obtained from the S1 barrel cortex of animals infected with E-SARE AAV. 0-150 ⁇ m from the surface.
  • FIG. 4B shows a representative example of two-photon live cell imaging obtained from cortical II / III layers after breeding mice infected with E-SARE AAV under three different conditions.
  • FIG. 4C shows the cumulative distribution of GFP intensity and GFP / RFP ratio collected from multiple animals and normalized for AAV infected neurons.
  • FIG. 5A shows the stimulation time course in the E-SARE reporter expression experiment.
  • FIG. 5B shows various promoter expression time courses using the absolute value of luciferase activity as an index.
  • FIG. 5C shows a time course plot of the data of FIG. 5B normalized to the 24-hour total average value for each time course. Error bars indicate standard error, *** is P ⁇ 0.001.
  • E-SARE AAV infected cells (RFP) and E-SARE activated cells (d2EGFP) are co-stained with the neuron-specific marker NeuN. The figure shows that E-SARE AAV infected cells (RFP) and E-SARE activated cells (d2EGFP) do not co-stain with the glial cell specific marker S100b.
  • FIG. 8 shows the results of examining the overlap of E-SARE-induced dGFP expression and endogenous Arc protein (a) or c-Fos protein (b) expression in somatosensory cortex of mice exposed to the new environment.
  • FIG. 9A shows the experimental procedure in which the induction of the E-SARE reporter in V1 by 10 minutes of light stimulation was examined.
  • FIG. 9B shows the V1 neuron image before and after the light stimulation (left), the quantification of the overlapping expression of E-SARE reporter dGFP, endogenous c-Fos and Arc (right) in the experiment of FIG. 9A.
  • FIG. 9C shows an image of E-SARE-dGFP and endogenous Arc immune activity (left) and quantification of the overlap of expression of E-SARE-dGFP and endogenous Arc (right) in the experiment of FIG. 9A.
  • FIG. 9D shows an image of E-SARE-dGFP and endogenous c-Fos immunoreactivity in the experiment of FIG. 9A (left), and quantification of overlap of E-SARE-dGFP and endogenous c-Fos expression (right).
  • FIG. 9E shows triple-stained images of E-SARE-dGFP, endogenous Arc protein, and endogenous c-Fos protein (upper) and quantification of expression overlap (lower) in the experiment of FIG. 9A.
  • Figure 10 shows the reconstructed 3D image of the AVV vector structure combining E-SARE and ER T2 CreER T2- PEST (left) and the axons of the labeled outer knee (LGN) projected onto the V1 layer 4 (Right).
  • FIG. 11A shows the degradation process of ER T2 CreER T2 , ER T2 CreER T2 -PEST protein and EGFP after treating cultured neurons with 20 ⁇ M anisomycin.
  • Figure 11B shows the results of quantitative examine the time course of ER T2 CreER T2, ER T2 CreER T2 -PEST protein and EGFP degradation.
  • One aspect of the DNA having a neural activity-dependent promoter activity according to the present invention is a CREB binding site, a MEF2 binding site, and an SRF binding among the synaptic activity responsive elements (SARE) on the promoter of the Arc / Arg3.1 gene.
  • the fragment containing the site comprises a construct linked in tandem at least twice.
  • RNA polymerase synthesizes (transcribes) mRNA based on DNA information.
  • “having a neural activity-dependent promoter activity” means that in a neuronal cell, when the neuronal cell is active, the promoter activity is exhibited and expression of a structural gene bound downstream is initiated, while the neuronal cell When is not active, it does not show promoter activity and does not initiate expression of the structural gene bound downstream.
  • a nerve cell is a cell constituting a nervous system composed of a central nerve (brain, spinal cord) and a peripheral nerve (somatic nerve, autonomic nerve).
  • Arc / Arg3.1 gene means a gene encoding activity-regulated cytoskeleton-associated protein.
  • the Arc / Arg3.1 gene is a member of the immediate early gene (IEG) family.
  • IEG immediate early gene
  • Many promoters and enhancer regions have been identified so far for the Arc / Arg3.1 gene, and SARE is one of them.
  • SARE region is located about 7kb upstream of the transcription start site, and transcription such as cyclic AMP response element-binding protein (CREB), myocyte enhancer factor 2 (MEF2), and serum response factor (SRF) Contains the binding site of the factor.
  • CREB cyclic AMP response element-binding protein
  • MEF2 myocyte enhancer factor 2
  • SRF serum response factor
  • One embodiment of the DNA having neural activity-dependent promoter activity according to the present invention includes a construct in which a fragment of SARE containing these transcription factor binding sites is linked in tandem at least twice.
  • the phrase “ligated in tandem at least twice” means that at least two identical fragments are linked in series. Fragments may be linked in the construct any number of times as long as they are more than once, for example, 3, 4, 5, 6, 7, 8, 9, or 10 times. Can do. Spacers may be inserted between the fragments as long as the resulting construct has promoter activity. Examples of the spacer include, but are not limited to, nucleic acids having a length of 1 to 20 bases, 2 to 15 bases, and 3 to 10 bases.
  • construct means an artificially produced DNA fragment.
  • the reporter gene linked downstream thereof has a high level depending on neural activity. It was confirmed that As shown in FIG. 1A, since this fragment is a highly conserved region between mammals, the DNA having a nerve activity-dependent promoter activity according to the present invention is similar in nerve activity-dependent in various mammalian cells. It is understood that the promoter activity is high.
  • the length of the SARE fragment containing three transcription factor binding sites can be, for example, between 50 bp and 200 bp, specifically 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 150 bp, 180 bp, As long as the obtained promoter has activity, it may be shorter or longer.
  • DNA having neural activity-dependent promoter activity includes a construct in which any one of the following DNAs (i) to (iv) is linked in tandem at least twice.
  • DNA consisting of the base sequence represented by SEQ ID NO: 2, 3, or 4 (ii) DNA consisting of a sequence in which one or more bases are deleted, added or substituted in the base sequence represented by SEQ ID NO: 2, 3, or 4; (iii) DNA comprising a sequence having 70% or more identity with the base sequence represented by SEQ ID NO: 2, 3, or 4; (iv) DNA capable of hybridizing under stringent conditions with a nucleic acid having a sequence complementary to the base sequence represented by SEQ ID NO: 2, 3, or 4.
  • sequences represented by SEQ ID NOs: 2, 3, and 4 are the base sequences of Fragment A, B, and C shown in FIG. 1A, respectively.
  • a reporter gene linked downstream thereof is expressed depending on neural activity, and such a reporter system. It was confirmed that the activity of neurons can be detected with high sensitivity and a wide dynamic range.
  • a promoter in which five Fragment A are linked has the highest expression level and a large dynamic range, and this was named E-SARE (SEQ ID NO: 1).
  • the DNA having neural activity-dependent promoter activity comprises a sequence in which one or more bases are deleted, added or substituted in the base sequence represented by SEQ ID NO: 2, 3, or 4. It may contain DNA.
  • the number of bases to be deleted, added or substituted is not particularly limited as long as the DNA has promoter activity, and can be, for example, 20 bases or less, 15 bases or less, 10 bases or less, 5 bases or less.
  • the DNA having a neural activity-dependent promoter activity may include DNA consisting of a sequence having 70% or more identity with the base sequence represented by SEQ ID NO: 2, 3, or 4. Good.
  • the identity may be 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher. Sequence identity can be determined according to known methods.
  • the DNA having neural activity-dependent promoter activity is a DNA that can hybridize under stringent conditions with a nucleic acid having a sequence complementary to the base sequence represented by SEQ ID NO: 2, 3, or 4. It may be.
  • Stringent conditions can be appropriately selected by those skilled in the art. For example, conditions such as 1 ⁇ SSC, 0.1% SDS, and 60 ° C. can be used. Stringency can be controlled by the concentration and temperature of SSC, and nucleic acids having higher complementarity can be obtained by selecting highly stringent conditions.
  • a spacer may be inserted between the DNAs (i) to (iv) linked in tandem as long as the obtained construct has promoter activity.
  • the spacer include, but are not limited to, nucleic acids having a length of 1 to 20 bases, 2 to 15 bases, and 3 to 10 bases.
  • the present invention also includes a vector (hereinafter simply referred to as “the vector according to the present invention”) comprising a DNA having the above-described neural activity-dependent promoter activity and a reporter gene linked downstream of the DNA.
  • the vector according to the present invention comprising a DNA having the above-described neural activity-dependent promoter activity and a reporter gene linked downstream of the DNA.
  • chloramphenicol acetyltransferase CAT
  • DsRed DsRed2, HcRed, HcRed1
  • green fluorescent protein GFP
  • RFP red Fluorescent protein
  • Venus Venus
  • ⁇ -glucuronidase GUS
  • ⁇ -galactosidase firefly or Renilla luciferase
  • blue fluorescent protein BFP
  • CFP cyan fluorescent protein
  • ECFP yellow fluorescent protein
  • YFP yellow fluorescent protein
  • genes encoding EYFP EYFP.
  • the vector according to the present invention can be prepared according to a known method.
  • the DNA and the reporter gene may be inserted into a commercially available reporter assay plasmid containing the necessary sequence in advance by a known method so that the DNA and the reporter gene are functionally linked.
  • “functionally bind” means that the DNA and the reporter gene are expressed so that the reporter gene is expressed when a transcription factor is bound to the DNA having neural activity-dependent promoter activity. Means that they are connected.
  • the vector according to the present invention can be introduced into a host cell by a calcium phosphate coprecipitation method, an electroporation method, a method of forming a complex with a polymer such as DEAE-dextran or polybrene.
  • the vector according to the present invention may be a viral vector. If a viral vector is used, the vector can be efficiently introduced into the target cell in vivo or in vitro by infecting the target cell.
  • a viral vector for example, a virus such as a retrovirus, adenovirus, adeno-associated virus vector, lentivirus, papilloma virus, vaccinia virus, baculovirus and the like can be used.
  • adeno-associated virus often infects cells that do not divide, so that vectors can be efficiently introduced into nerve cells.
  • it is non-pathogenic, does not cause overexpression or deletion of endogenous proteins, and is not integrated into the genome, so it is preferably used in the present invention as a safe and versatile vector.
  • an expression cassette for an infectious marker gene When using a viral vector, it is also preferable to insert an expression cassette for an infectious marker gene into the vector. Thereby, the expression of the infection marker gene can be confirmed, and it can be evaluated whether or not the vector has been introduced into the target cell.
  • the infection marker gene is not particularly limited as long as its expression can be easily confirmed, and any of the reporter genes described above may be used.
  • a promoter for expressing an infection marker gene a promoter that is highly active in nerve cells and not responsive to nerve activity is preferable. Examples of such a promoter include the E-PGK promoter (SEQ ID NO: 5) described in Examples.
  • the E-PGK promoter is a PGK-promoter fused with an intron fragment.
  • the vector By inserting a reporter gene driven by DNA having neural activity-dependent promoter activity and an infection marker gene that expresses a marker protein distinguishable from the reporter gene product into the vector according to the present invention, the vector It can be confirmed at the single cell level at the same time whether the nerve cell has been introduced and the presence or absence of the activity of the nerve cell.
  • the present invention also includes host cells containing the vector according to the present invention (except for cells in a human body).
  • Host cells include cells in vivo from any non-human animal and cultured cells from animals including humans.
  • the present invention also includes a non-human animal comprising a vector into which a DNA having neural activity-dependent promoter activity according to the present invention is inserted.
  • Such animals can be, for example, mice, rats, rabbits, dogs, monkeys, sheep, cows, horses, etc., and can be used for various tests.
  • the vector according to the present invention can be used for the following purposes, for example.
  • the nerve cell activity detection method comprises: Transfecting neurons with the vector according to the present invention; Measuring reporter gene expression to identify activated neurons.
  • the nerve cell may be a cell in a living body or a cultured cell.
  • the cells in the living body are preferably non-human animal cells. After the transfection, the nerve cells may be stimulated, and the neurons that have acted in response to the stimulation may be identified, or the activity due to spontaneous firing of the neurons may be identified.
  • the step of stimulating nerve cells can be performed according to a known method. For example, in the case of cells in vivo, the animal may be stimulated from the outside, or the cultured cells may be directly electrically or chemically stimulated. May be given.
  • the expression of the reporter gene can be measured by a known method depending on the type of the reporter gene, whereby the presence / absence or strength of nerve cell activity can be easily evaluated.
  • the method for evaluating differentiation of nerve cells according to the present invention includes: Transfecting a cell according to the present invention into a cell at any stage of differentiation from a stem cell to a nerve cell; Measuring the expression of the reporter gene and evaluating the presence or absence or intensity of neural activity.
  • Stem cells include, but are not limited to, neural stem cells, iPS cells, ES cells and the like.
  • nerve plasticity can be detected at the cell level.
  • the structure and function of the nervous system are not necessarily fixed, but change depending on the state of activity. This characteristic is called neuroplasticity. Changes due to neural plasticity occur at various levels, such as individual synapse level, cell level, neural circuit level, individual level, and contribute to learning, memory, recovery from brain damage, and the like. It is considered that one of the mechanisms of neuroplasticity is the change in spinous process morphology depending on neural activity, and the formation and disappearance of synapses themselves.
  • the method for evaluating differentiation of nerve cells according to the present invention includes: Transfecting a cell according to the present invention into a cell at any stage of differentiation from a stem cell to a nerve cell; Measuring the expression of the reporter gene and evaluating the presence or absence or intensity of neural activity.
  • the vector according to the present invention can detect the survival or death of nerve cells in real time and evaluate the presence or absence of brain function. For example, during surgery such as cerebral infarction, trauma, and hematoma removal, the vector according to the present invention is introduced into a neuron as a brain function marker, and the expression of a reporter gene is detected to determine whether the cell is alive or dead. It can also be excised.
  • the efficacy and safety of drug candidate compounds such as brain function improving agents and neuroprotective agents can be evaluated.
  • a method is Transfecting neurons with the vector according to the present invention; Administering a candidate compound to a nerve cell; Measuring the expression of the reporter gene to assess the presence or absence of neural activity.
  • the nerve cell may be a cell in a living body or a cultured cell. In the case of in vivo cells, non-human animal cells are preferred.
  • a candidate compound and its administration method can be appropriately selected by those skilled in the art.
  • the drug candidate compound a low molecular compound, a high molecular compound, a nucleic acid, a protein and the like can be used.
  • the expression of a reporter gene is increased by administration of a drug candidate compound, it can be evaluated that the compound has the potential to improve brain function, and when the expression of the reporter gene is decreased, it is toxic to neurons. Can be evaluated.
  • luciferase reporter plasmids used were constructed on pGL4.11-luc2P firefly luciferase reporter plasmid (Promega).
  • the E-SARE (5xSARE-ArcMin) promoter is constructed by linking five SARE-ArcMin (Non-patent Document 7) 104 bp SARE enhancer fragments (-6793 to -6690 when the transcription start site is +1). did.
  • the E-PGK promoter was constructed by fusing a mouse PGK promoter fragment (-280 to +112) with an intron fragment (+69 to +315) of human growth hormone (GH).
  • the mouse c-fos promoter (-622 to +140) was obtained from pGEM4-cFos-LacZ (non-patent document 2; provided by Dr. Tom Curran).
  • the 5xCRE-minCMV promoter (SEQ ID NO: 6), 5xMRE-minCMV promoter (SEQ ID NO: 7), and 5xSRE-minCMV promoter (SEQ ID NO: 8) are respectively represented by 5xCRE (CRB binding site TGACGTCA (SEQ ID NO: 11) and 5 ⁇ MRE (MEF2 binding site CTATTTTTAG (SEQ ID NO: 12) and 5 repeats of 7 bp spacer), and 5 ⁇ SRE (SRF and Elk-1 binding site AGGATxxCCATATTAGG (SEQ ID NO: 13) and 6 bp And the minimal CMV promoter fragment (-51 to +6; SEQ ID NO: 9)).
  • the d2EGFP reporter plasmid was constructed by transferring the promoter fragment from the luciferase plasmid to pGL4.11-d2EGFP (T. Kawashima et al., Proc Natl Acad Sci USA 106 (1), 316 (2009).). The numbers indicating the position on the genome in the above are based on the NCBI genome database.
  • the E-SARE double expression cassette (SEQ ID NO: 10) was made from the following elements: LVDP lentiviral plasmid (J. Tian and ST Andreadis, Gene Ther 16 (7), 874 (2009) .; Dr.
  • Stelios CHS4 insulator element (provided by Andreadis); WPRE element of CSII lentiviral plasmid (provided by Dr. Hiroyuki Miyoshi); TurboFP635 of pTurboFP635-C (Evrogen); pCS2-Venus (T. Nagai) et al., Nat Biotechnol 20 (1), 87 (2002); provided by Dr. Atsushi Miyawaki.) Venus; pGL4.11 (Promega) SV40 polyA and pcDNA3 (Invitrogen) cattle GH polyA.
  • the prepared cassette was inserted into the AAV plasmid pFBGR (provided by Dr. Beverly Davidson and Dr.
  • d2Venus was prepared by fusing the Venus and pd2EGFP-1 (Clontech) MODC PEST sequences.
  • a blue-type E-SARE double expression cassette for immunohistochemical analysis was prepared by replacing d2Venus with d2EGFP and TurboFP635 with TagRFP (Evrogen).
  • Luciferase and d2EGFP reporter assays were performed using cultured cortical neurons obtained from Sprague-Dawley rat embryos. Virus administration experiments were performed on male C57BL / 6N mice, 4-10 weeks old. Wild type mice were purchased from Japan SLC, Inc.
  • Luciferase and d2EGFP assay in cultured neurons were performed according to a known method (Non-patent Document 7). Briefly, rat E18 embryo cortex was first transfected with a luciferase plasmid and Nucleofector (Lonza). Neurons were silenced with 1 ⁇ M tetrodotoxin (TTX) at DIV (days in vitro) 10 days. Nerve cells were stimulated with DIV11 in a medium (4AP & BIC medium) containing 100 ⁇ M 4-AP, 30 ⁇ M bicuculline, 100 ⁇ M glycine, and 1 ⁇ M strychnine for 4 hours.
  • TTX tetrodotoxin
  • the luciferase activity of the cell lysate was measured with the Dual-Luciferase Reporter Assay System (Promega).
  • d2EGFP reporter assay 3.75 ⁇ 10 6 cultured neurons were transfected with d2EGFP reporter plasmid (3.6 ⁇ g) and transfection marker RFP plasmid (0.9 ⁇ g) as described above. Nerve cells were silenced as described above, then stimulated and then fixed with paraformaldehyde.
  • fireporate reporter plasmid (4.8 ⁇ g) and pGL4.74 Renilla control plasmid (1.2 ⁇ g) were electroporated into 1.0 ⁇ 10 7 cortical neurons, and cell suspensions in 24 wells Were evenly dispensed into 8 wells of a poly D lysine-coated plate. Nerve cells were silenced with 1 ⁇ M TTX at DIV10 and stimulated with DIV11 for 10 minutes in 4AP & BIC medium or Mg 2+ free Tyrode solution containing 10 ⁇ M glutamate for 2 minutes.
  • the medium was changed twice at 5-minute intervals during 10-minute stimulation to eliminate antagonism by residual TTX.
  • the neurons were again silenced in a medium containing 1 ⁇ M TTX, 20 ⁇ M NBQX, and 50 ⁇ M D-AP5.
  • Cell lysates are collected from one well each at predetermined time points (0, 2, 4, 6, 8, 12, 16 and 24 hours after silent stimulation) and stored at -80 ° C until measurement of luciferase activity did.
  • T Transcription
  • R mRNA level
  • P protein level
  • D mRNA and D protein indicate the attenuation constants of mRNA and reporter protein, respectively.
  • D mRNA consists of mRNA decay constant of mRNA without intron (HF Wang, L. Feng, and DK Niu, Biochem Biophys Res Commun 354 (1), 203 (2007).) And mRNA decay constant of similar luciferase vector ( X. Fan, E. Roy, L. Zhu et al., J Biol Chem 278 (12), 10232 (2003).) (0.421 / h).
  • D protein was set to a value (0.641 / h) obtained by measurement with cultured neurons (data not shown).
  • the measured temporal data was interpolated and numerically differentiated to obtain an outline of the transcription response. Based on this shape, it was assumed that there are two phases in the transcriptional response to the stimulus.
  • the first phase is a rectangular pulse-like transfer that lasts 1-2 hours (or less) and the second phase is a slowly decreasing linear transfer that takes several hours or more to return to zero. is there.
  • the first phase was defined by the pulse height (a1) and length (t1), and the second phase was defined by the initial height (a2) and time to return to zero (t2).
  • Maximum likelihood values of these parameters were determined by MLE.
  • the likelihood of each parameter was calculated by the following formula.
  • G is the Gaussian probability density
  • M i is the simulation data based on the parameters a1, a2, t1, t2 of each time point
  • D i and ⁇ i are the average of the measured values of the luciferase data at time point i
  • N is the number of measured time points.
  • AAV adeno-associated virus
  • Serotype 1 adeno-associated virus is produced in the baculovirus / Sf9 expression system (developed by Robert Kotin's team with the assistance of Beverly Davidson's team). Purified. Specifically, first, ADH plasmid and RepCap1 helper plasmid (M. Urabe, C. Ding, and RM Kotin, Hum Gene Ther 13 (16), 1935 (2002).) Were used for DH10-Bac E. coli (Invitrogen). ) To produce a recombinant baculovirus bacmid.
  • the baculovirus titer uses a bacmid specific primer (e56-F: 5'-AATGATAGGCATTAACTTGC-3 '(SEQ ID NO: 16); e56-R: 5'-GTGATTTAGTTGGCGACTTG-3' (SEQ ID NO: 17)). And measured by quantitative PCR.
  • AAV was produced by the following method.
  • hypotonic lysis buffer 1% Triton X-100, 10 mM HEPES (pH 8.0), 3 mM NaCl, and 0.5 mM MgCl 2
  • the lysate was centrifuged, and the supernatant was mixed with AVB Sepharose (GE Healthcare) and placed at room temperature for 1 hour. The mixture was passed through a gravity-flow column (Bio-Rad) and then washed with PBS. Elution was carried out with an elution buffer (50 mM glycine (pH 3.0)), centrifuged using Amicon Ultra 100K Centrifugal concentrators (EMD Millipore), and further concentrated. During the concentration process, the virus buffer was replaced with PBS. When measured by quantitative PCR, the resulting virus titer was usually 0.5 to 3.0 ⁇ 10 13 genome copies / ml (total volume 200 ⁇ l).
  • Virus-administered mice by stereotaxic fixation The virus was administered by stereotaxic fixation and a stretched glass micropipette. Mice were pretreated with 2.5 mg / kg atropine sulfate (Tanabe Pharma) and anesthetized by intraperitoneal injection of 440 mg / kg chloral hydrate (Tokyo Chemical Industry). Petrolatum was applied to both eyes to prevent drying and the scalp was treated with a hair removal cream. The mouse was fixed to a stereotaxic apparatus for small animals (David Kopf Instruments). The scalp was incised along the midline, and the periosteum was removed with 3% hydrogen peroxide and a scalpel.
  • the skull was thinned with a drill and a small hole was made with a 27 gauge needle.
  • a stretched micropipette was connected to a Hamilton syringe (Hamilton Company), and the virus was injected.
  • the Hamilton syringe was connected to a syringe pump (World Precision Instruments).
  • the micropipette was tilted to 45 degrees.
  • the stereotaxic administration was performed on the following tissues according to the following coordinates.
  • Visual cortex A / P -3.4 mm from bregma, M / L ⁇ 3.0 mm, D / V -0.5 mm from buffy coat surface.
  • Somatosensory Barrel Cortex A / P -1.5 mm from bregma, M / L ⁇ 3.0 mm, D / V -0.5 mm from buffy coat surface.
  • the virus solution was injected at 1.0 to 1.5 ⁇ l at a rate of 0.05 to 0.1 ⁇ l / min, and after administration, the pipette was fixed in place for 10 minutes and then removed. After removing the micropipette, the incised skin was sutured, antibiotic cream was applied, and an analgesic was administered subcutaneously to ease postoperative pain. After administration, the animals were bred as usual for 4 weeks to 2 months until they were used in the experiment.
  • E-SARE induction experiment in visual cortex Mice administered with E-SARE AAV in the visual cortex were left in a dark environment for 2 days after the removal of a single eye. On the day of the experiment, the mice were exposed to the light environment for 4 hours and then fixed by transcardial perfusion. The brain was removed and fixed and antifreeze treated according to a known method (Non-Patent Document 7). The macroscopic fluorescence image of the brain was taken with a digital CMOS camera (Panasonic) mounted on a stereomicroscope (Olympus). Coronal sections (thickness 50 ⁇ m) of the brain were prepared with a cryostat microtome (Carl Zeiss) and observed with a fluorescence digital microscope (KEYENCE).
  • Immunohistochemistry For immunohistochemical analysis, a discolored E-SARE AAV was used in which d2EGFP was expressed under the E-SARE promoter and TagRFP was expressed under the E-PGK promoter. The fluorescence spectra of EGFP and TagRFP allowed the use of an additional far-red channel to stain cell type specific markers.
  • the immunohistochemical analysis was performed according to a known method (Non-patent Document 7).
  • a 50 ⁇ m-thick brain slice was treated with 0.3% Triton X-100 / PBS for 5 minutes to make it permeabilized, and blocking solution (0.1% Triton X-100, 1% bovine albumin, and 5% normal goat serum in PBS ) At room temperature for 2 hours. Subsequently, the sections were reacted with a primary antibody diluted with a blocking solution at 4 ° C. for 2 hours.
  • Primary antibodies include mouse anti-Arc monoclonal antibody (C-7, 1: 250; Santa Cruz), rabbit anti-RFP polyclonal antibody (R10367, 1: 1000; Invitrogen), mouse anti-GFAP monoclonal antibody (clone GA-5, 1: 1000; Sigma), mouse anti-S100 ⁇ monoclonal antibody (clone 4C4.9, 1: 500; Abcam), and mouse anti-NeuN monoclonal antibody (clone A60, 1: 1000; Chemicon).
  • a secondary antibody diluted with a secondary antibody solution (0.1% Triton X-100 and 5% normal goat serum in PBS) at room temperature for 2 hours.
  • Secondary antibodies included Alexa647-conjugated goat anti-mouse IgG antibody and Alexa555-conjugated goat anti-rabbit IgG antibody (1: 500, Invitrogen).
  • the immunostained sections were further stained with Hoechst 33342 and mounted on tissue glass slides. Fluorescence images were obtained with an LSM510 confocal microscope (Carl Zeiss) or A1R confocal microscope (Nikon).
  • TR group A group with a trimmed beard
  • HC group a group placed in a normal cage
  • NE group a group placed in a new environment
  • TR group mice had their beard trimmed 2 days prior to imaging and left in the dark until imaging.
  • HC group mice were bred as usual until imaging.
  • NE group mice were placed in a dark environment for 2 days, then moved to a new environment composed of various objects, and imaged 6-7 hours later.
  • the infection site was confirmed by detecting the infection marker RFP signal that penetrated the skin and skull with a portable fluorescence observation system (RelyOn).
  • 3D stacking image (XY width: 485 ⁇ m, Z depth: 400 ⁇ m, Z depth interval: 3 ⁇ m) is the mode lock pulse Ti of LSM 7MP two-photon microscope (Carl Zeiss) and Mai Tai DeepSee (Spectra-Physics): Obtained using a combination of Sapphire lasers (900 nm). 3D volume rendering was performed with Imaris software (Bitplange). Analysis of E-SARE reporter expression was performed using custom Matlab software (Mathworks) offline.
  • Infected neurons were identified using marker RFP expression with a custom algorithm. The cell recognition accuracy exceeded 98%, and more than 80% of the image stack could be identified. Typically, 1500-2500 cells were identified from the II / III layer of a single stack in each mouse. The GFP and RFP fluorescence of each cell was calculated from the average of the fluorescence intensity at the center of the cell (4.5 ⁇ mx 4.5 ⁇ m). Comparison of E-SARE reporter expression levels under different conditions was performed as follows. First, it was assumed that 10% or more of the cell group in each stack was non-responsive cells. Previous studies have shown that the primary visual cortex contains silent cells that do not respond to sensory stimuli, and silent cells represent more than 10% of all neurons (DH O'Connor, SP Peron , D.
  • the distribution of cell soma fluorescence of the entire cell group can be normalized by the distribution of cell soma fluorescence of non-responsive cells (for example, the lower 10% of the cell group).
  • the differences in the normalized distribution between each group of mice can then be compared.
  • Each stack was first divided into Z sublayers with a thickness of 30 ⁇ m. Each sublayer usually contained 100-300 cells. Cellular GFP and GFP / RFP fluorescence signals were normalized within each sublayer relative to the lower 10% of cells in each sublayer. Standardized values were collected for each mouse, and then averaged between mice under the same conditions.
  • E-SARE had the highest expression level, which was named E-SARE and used in the subsequent experiments.
  • E-SARE had a luciferase expression level of 100 or more higher than that of a reporter plasmid using a promoter in which five SARE transcription factor binding sites (CREB, MEF2, and SRF) were linked (FIG. 1C).
  • E-SARE has at least 30-fold higher activity-dependent expression level and 20-fold wider dynamic range than the c-fos promoter, the most active promoter reported to date (Fig. 1D).
  • d2EGFP destabilized EGFP
  • E-SARE produced significantly stronger fluorescence than when the c-Fos promoter was used (FIG. 1E).
  • the E-SARE promoter is expressed in the same mechanism as the endogenous Arc promoter induces genes in vivo. It was strongly suggested to induce The E-SARE AAV vector is a safe and versatile cell labeling tool because it does not cause overexpression or deletion of endogenous proteins and is not integrated into the genome. The persistence of E-SARE reporter expression can be very important for anatomical and electrophysiological analysis after live imaging as well as for imaging large numbers of highly responsive cells in vivo or in vitro. Useful.
  • AAV associated virus
  • the virus is composed of two expression cassettes separated by an insulator (J. Tian and S. T. Andreadis, Gene Ther 16 (7), 874 (2009) .; cHS4 in FIG. 3A).
  • the first is a neural activity-dependent reporter cassette in which d2Venus (GFP with a half-life of 2 hours) is driven by E-SARE, and the second is an E-PGK promoter in which TurboFP635 (RFP) is insensitive to neural activity.
  • E-PGK insensitive PGK promoter
  • mice that had been injected with E-SARE AAV were previously established Arc Induction experiments were applied.
  • the primary visual cortex (V1) on both sides of the mouse was injected with a reporter virus, and after one month, one eye was removed and placed in the dark for 2 days, and then exposed to a bright environment for 4 hours (FIG. 3B).
  • GFP fluorescence in the cortex on the opposite side was significantly increased compared to the cortex on the same side as the open eye.
  • RFP fluorescence was comparable in both cortex ( Figure 3E).
  • Barrel cortex of somatosensory cortex is well known as an area containing various types of nerve cells that fire independently, as an E-SARE driven reporter as a marker of information code neurons in the barrel cortex.
  • the barrel cortex is a cortical region that develops in animals with a beard and converts the deflection when the beard contacts an object into information.
  • DH O'Connor, SP Peron, D Huber et al., Neuron 67 (6), 1048 only a small group of cells in the barrel cortex respond strongly to whiskers activity. It is shown that there is. Therefore, it is very difficult to identify neuronal activity and electrophysiological recording by whisker stimulation.
  • E-SARE with an expanded dynamic range can selectively visualize highly active neurons, or is E-SARE useful for analyzing this complex electrical circuit during in vivo electrical recording?
  • Fig. 4A In vivo live imaging of E-SARE reporter-expressing mice by two-photon microscopy successfully infected thousands of II / III neurons in the barrel cortex and successfully imaged the reporter virus (Fig. 4A).
  • TR trimmed beards
  • HC familiar cage
  • NE unshaved mice exposed to the new environment
  • the distribution of cells expressing GFP was increased in descending order of the amount of whiskers used (NE, HC, TR) (FIGS. 4B and 4C).
  • FIGS. 8A and 8B show representative images of tissue sections. Scale bar is 50 ⁇ m. Arrows indicate neurons that were positive for both E-SARE-dGFP and Arc / c-Fos, and triangles were neurons that were positive for only one (dGFP image is a neuron that expressed only dGFP) In the Arc / c-Fos image, nerve cells expressing only the Arc / c-Fos protein are shown. The lower part of FIGS.
  • FIG. 9A shows the experimental procedure. 7-8 week old mice were infected with E-SARE AAV, after 3-4 weeks, placed in dark environment for 2 days, exposed to light environment for 10 minutes, and placed in dark environment again 90 minutes later It was. The V1 neuron image before and after the light stimulus is shown on the left of FIG. 9B.
  • FIG. 9B right shows the quantification of immune activity of E-SARE reporter dGFP and endogenous c-Fos and Arc. Error bars represent standard error in 4 mice each. * P ⁇ 0.01. *** is P ⁇ 0.001 (unpaired t-test).
  • FIGS. 9C and D show the overlapping expression of E-SARE-dGFP and endogenous Arc / c-Fos protein.
  • E-SARE-dGFP images of E-SARE-dGFP, the infection marker RFP, and the endogenous Arc / c-Fos immune activity are shown.
  • Arrows indicate cells positive for both E-SARE-dGFP and Arc / c-Fos
  • triangles indicate cells positive for either E-SARE-dGFP or Arc / c-Fos .
  • FIG. 9E shows triple staining of E-SARE-dGFP, endogenous Arc protein, and endogenous c-Fos protein.
  • the upper panel shows E-SARE-dGFP and the immune response of endogenous Arc protein and endogenous c-Fos protein.
  • the arrows indicate neurons that are positive for all three of E-SARE-dGFP, endogenous Arc protein, and endogenous c-Fos protein.
  • the quantification of the overlap is shown in the lower row.
  • Numbers in parentheses indicate the number of cells counted in 4 animals.
  • FIG. 10 shows a reconstructed 3D image of the LGN axon projection on V1 (scale bar: upper 20 ⁇ m, lower 5 ⁇ m)
  • Layer 4 depth -340 ⁇ m, plane indicated by a straight line
  • E-SARE-inducible Cre recombinase is expressed in cells activated at the time of single eye stimulation, and cells that are activated only at cells that respond to input by single eye stimulation and RFP is activated at a specific time. Only shows that the projection axons to V1 were stained. That is, it was shown that E-SARE expresses an arbitrary gene only in a cell group having a history of specific neural activity in a cell population having other identical molecular characteristics.
  • FIG. 11 shows the results of examining the protein half-life of the destabilizing drug-induced recombinase ER T2 CreER T2- PEST.
  • FIG. 11A shows the degradation process of ER T2 CreER T2 and ER T2 CreER T2- PEST protein after treating cultured neurons with 20 ⁇ M anisomycin. Both ER T2 CreER T2 and ER T2 CreER T2- PEST proteins (arrows) were gradually degraded after anisomycin treatment, but the EGFP protein in the same sample was hardly degraded.
  • FIG. 11B shows the results of quantitative examination of the degradation over time. The half-life of ER T2 CreER T2- PEST used in FIG. 10 was calculated to be 2.9 hours. Error bars indicate standard error in three independent experiments.
  • SEQ ID NO: 1 represents the base sequence of the E-SARE promoter.
  • SEQ ID NO: 2 represents the base sequence of Fragment A shown in FIG. 1A.
  • SEQ ID NO: 3 represents the base sequence of Fragment B shown in FIG. 1A.
  • SEQ ID NO: 4 represents the base sequence of Fragment C shown in FIG. 1A.
  • SEQ ID NO: 5 represents the base sequence of the E-PGK promoter.
  • SEQ ID NO: 6 represents the base sequence of 5xCRE-minCMV promoter.
  • SEQ ID NO: 7 represents the nucleotide sequence of 5xMRE-minCMV promoter.
  • SEQ ID NO: 8 represents the base sequence of 5xSRE-minCMV promoter.
  • SEQ ID NO: 9 represents the base sequence of the minimum CMV promoter fragment minCMV.
  • SEQ ID NO: 10 represents the base sequence of the E-SARE double expression cassette.
  • SEQ ID NO: 11 represents the nucleotide sequence of the CRB binding site used for the 5xCRE-minCMV promoter.
  • SEQ ID NO: 12 represents the base sequence of the MEF2 binding site used for the 5xMRE-minCMV promoter.
  • SEQ ID NO: 13 represents the nucleotide sequence of the SRF and Elk-1 binding site used for the 5xSRE-minCMV promoter.
  • SEQ ID NO: 14 represents the nucleotide sequence of M13 forward primer.
  • SEQ ID NO: 15 represents the nucleotide sequence of M13 reverse primer.
  • SEQ ID NO: 16 represents the base sequence of the e56 forward primer.
  • SEQ ID NO: 17 represents the base sequence of the e56 reverse primer.

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Abstract

L'invention a pour objectif de fournir un promoteur artificiel qui génère une action de manière dépendante par rapport à l'activité nerveuse, et qui présente une haute sensibilité et une plage dynamique importante, et de fournir un procédé, ou similaire, destiné à identifier une région d'activité nerveuse en mettant en œuvre ledit promoteur. Plus précisément, l'invention concerne un ADN possédant une activité promotrice dépendante de l'activité nerveuse dans lequel un fragment incluant une région de liaison CREB, une région de liaison MEF2 et une région de liaison SRF, dans un élément sensible à l'activité des synapses (SARE) présents sur un promoteur d'un gène Arc/Arg3.1, contient une construction connectée au moins en tandem double.
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WO2022040432A1 (fr) * 2020-08-19 2022-02-24 Stridebio, Inc. Vecteurs de type virus adéno-associé pour le traitement du syndrome de rett

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