The invention provides transgenic mice comprising tau transgenes, and methods of preparing and using the transgenic mice. For example, the invention provides a transgenic mouse, the genome of the cells of which stably comprise a DNA molecule which comprises a human genomic DNA sequence comprising a human tau promoter and which DNA sequence encodes human tau.

BACKGROUND OF THE INVENTION

The human tau protein has been implicated in the pathogenesis of several human neurodegenerative diseases including Alzheimer's disease (AD) and frontal temporal lobe dementia (Hardy et al. 1998, Spillantini and Godert 1998). The pathology of AD is defined as the presence of amyloid-containing plaques and neurofibrillary tangles (NFTs) composed of tau arranged into paired helical filaments (PHFs). Mutations in the tau gene lead to a range of tauopathies (termed Fronto-Temporal Dementia and Parkinsonism linked to chromosome 17 (FTDP-17) where tau takes the form of PHF (Spillantini et al. 1996, Poorkaj et al. 1998, Hutton et al. 1998) or twisted ribbons (Spillantini et al. 1997, 1998, Hutton et al. 1998, Reed et al. 1998). Although the mechanisms underlying the development of tauopathy in these diseases are unknown, hyperphosphorylation of tau has been linked to AD (Iqbal and Iqbal 1996), and disruption of microtubule binding and assembly has been linked to FTDP-17 missense mutations (Hasegawa et al. 1998, Hong et al. 1998).

Human tau is alternatively spliced to generate six isoforms that differ in the presence of absence of exons 2, 3 or 10 (Godert et al. 1989, Andreadis et al. 1992). Splicing out of exon 10 generates a tau protein with 3 microtubule binding domain repeats (3R), whereas its inclusion generates tau with 4 repeats (4R). The normal human brain maintains an approximately equal ratio of 4R to 3R tau but this ratio is shifted in favor of more 4R tau in FTDP-17 patients with splice site mutations (Godert and Jakes 1990, Spillantini et al. 1998, Hong et al. 1998, Godert et al. 1999, Grover et al. 1999). Biochemical evidence suggests that microtubule binding and assembly is disrupted by some missense mutations in tau (Hasegawa et al. 1998, Hong et al. 1998, Dayanandan et al. 1999), however, the mechanism by which excess 4R tau causes neuronal degeneration is less clear. Given that excess 4R tau is detrimental to humans, it is surprising that the normal adult mouse makes 4R tau exclusively (Gotz et al. 1995, Kampers et al. 1999), although it is possible that in FTDP-17, a shift in the normal ratio of tau isoforms is pathogenic rather than their absolute levels.

Thus, what is needed is a non-human animal model which expresses human tau, e.g., to examine the normal biology of tau and to provide a model for tauopathies where the ratio of tau isoforms are shifted.

SUMMARY OF THE INVENTION

The invention provides a transgenic rodent, e.g., a rat or a mouse, the genome of the cells of which are augmented with a human genomic DNA sequence encoding tau. Preferably, the expression of the human genomic DNA sequence results in the presence of at least one, and preferably two or more, e.g., all six, isoforms of human tau. It is envisioned that the human genomic DNA sequence comprises wild-type human tau sequences, as well as sequences which have alterations, e.g., deletions, insertions or mutations, for example, a splice site or missense mutation, e.g., one encoding an amino acid substitution, such as those which result in a shift in the ratio of the isoforms. Preferably, the alterations yield tau protein that is associated with dementing disorders, including neurodegenerative disorders, in humans. As described hereinbelow, to examine the normal cellular function of tau and its role in pathogenesis, transgenic mice were prepared that over-express a tau transgene derived from a human PAC that contains the coding sequence, intronic regions and regulatory regions of the human gene. All six isoforms of human tau are represented in the transgenic mouse brain at the mRNA and protein level and the human tau is distributed in neurites and at synapses, but is absent from cell bodies. A comparison between the genomic tau mice and transgenic mice that over-express a tau cDNA transgene shows that overall, the distribution of tau is similar in the two lines, but human tau is located in the somatodendritic compartment of many neurons in the cDNA mice. Tau-immunoreactive axonal swellings were found in the spinal cords of the tau cDNA mice, which correlated with a hind-limb abnormality whereas neuropathology was essentially normal in the tau genomic DNA mice up to eight months of age. Further provided are progeny of human genomic tau mice, e.g., progeny of a cross between genomic tau mice and tau knock out mice, e.g., a C129/C57B16 knock out. As described hereinbelow, such progeny mice had somatodendritic localization of tau which was in an abnormal conformation. Thus, in one embodiment of the invention, the human genomic tau DNA mice do not express murine tau, e.g., due to a knock out of the endogenous murine tau gene(s). Onset of abnormal pathologies in tau transgenic mice can occur at any time in development. For example, in one embodiment of the invention, the human genomic tau mice present a normal phenotype early in life; however, during the course of development, the human tau genomic mice develop one or more abnormal pathologies, e.g., motor and/or neurological pathologies. In another embodiment, abnormal pathologies may be present before or immediately after birth and optionally those pathologies progressively worsen.

The invention also provides a method of preparing a transgenic rodent of the invention. The method comprises contacting a rodent cell which can give rise to an organism, e.g., a totipotent cell such as a fertilized embryo, with an isolated and purified DNA molecule comprising a human genomic DNA sequence encoding tau so as to yield a transformed cell. The transformed cell is manipulated so as to yield a rodent. Then it is determined whether the rodent comprises cells comprising the human genomic DNA sequence encoding tau, i.e., it is a transgenic rodent. Preferably, the rodent expresses human tau. For example, the rodent may express all six forms of human tau, a subset of the human isoforms, and/or an altered ratio of the human isoforms. The expression of a subset of isoforms, e.g., one or more but less than all six of the human isoforms, or an altered ratio of human isoforms, may be the result of one or more alterations in the human genomic DNA sequence relative to a sequence, the expression of which results in the presence of all six human isoforms in the rodent.

Further provided is an isolated and purified DNA molecule comprising a human genomic DNA sequence encoding tau.

Also provided is a method to employ the rodent of the invention, e.g., to screen for an agent that inhibits or reduces neurodegeneration or tauopathies, e.g., such as those which are associated with Alzheimer's disease, frontal temporal lobe dementia, FTPD-17, and the like.

The invention also provides a transgenic rodent comprising a humanized tau gene. For example, an isolated and purified murine DNA sequence encoding tau is altered so that at least one, preferably at least one fourth, and more preferably at least a majority, of the codons in the murine tau coding region are humanized, thus, providing a humanized murine tau DNA sequence. The humanized murine tau DNA sequence may encode native murine tau or may comprise alterations, i.e., insertions, deletions, or mutations, e.g, which result in amino acid substitutions, or any combination thereof. Then the isolated and purified humanized murine tau DNA sequence is introduced into a murine cell that can give rise to an organism, e.g., a totipotent cell such as a fertilized embryo, so as to yield a transformed murine cell. The transformed cell is manipulated so as to yield a mouse. Then it is determined whether the mouse comprises cells comprising the humanized murine tau DNA sequence, i.e., it is a transgenic mouse. Preferably, the transgenic mouse of this embodiment of the invention expresses the humanized tau. More preferably, the expression of the humanized tau in the transgenic mouse is associated with the development of a pathology in the mouse.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a transgenic rodent comprising a tau transgene, e.g., a humanized rodent tau transgene or a human genomic tau transgene, and methods of preparing and using the transgenic rodents. The transgenic rodents are prepared with an isolated and purified DNA sequence encoding tau. The isolated and purified DNA sequence which encodes tau may represent native sequences, including tau sequences that are not associated with any pathology in humans and tau sequences that are associated with pathology in humans, or may represent novel sequences, e.g., humanized murine tau sequences. The expression of the DNA sequence in the transgenic rodent results in tau protein. The transgenic rodents of the invention are useful to test agents, e.g., agents that inhibit or prevent pathological neurodegeneration, to determine which environmental or genetic factors other than tau predispose an organism to a dementing disorder, or as a source of tau, e.g., to prepare antibodies.
 

Claim 1 of 6 Claims

1. A transgenic mouse, the genome of the cells of which stably comprise a DNA molecule which comprises a human genomic DNA sequence comprising a human tau promoter and which DNA sequence encodes human tau, wherein the DNA sequence is expressed in the transgenic mouse so as to result in the transgenic mouse expressing six isoforms of human tau, wherein the transgenic mouse does not express endogenous murine tau, wherein the DNA sequence comprises one SrfI restriction site in the human tau coding region, and wherein the transgenic mouse accumulates filamentous tau in dendrites of hippocampal neurons.

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