Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
1998-05-14
2002-09-17
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C435S029000, C435S354000
Reexamination Certificate
active
06452065
ABSTRACT:
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates to a transgenic non-human animal lacking native presenilin 1 (PS1) protein and a transgenic non-human animal expressing either the wild-type human PS1 or human PS1 containing a Familial Alzheimer's Disease (FAD) mutation on native PS1 null background. The transgenic animal can be used in the study of the in vivo functions of PS1 and the effect of FAD mutation in PS1 function both during embryonic development and during aging. The transgenic animal can also be used in the identification of compounds that modulate the expression or activity of PS1.
BACKGROUND OF THE INVENTION
Presenilin 1 (PS1) is a protein expressed in the central nervous system as well as other tissues of animals from early embryonic development through adult life. Endoproteolytically processed in vivo, (G. Thinakaran et al., Neuron 17:181 (1996)), PS1 is an eight transmembrane protein homologous to sel-12, a
C. elegans
protein that facilitates signaling mediated by the Notch/in-12 family of receptors (D. Levitan & I. Greenwald, Nature, 377:351 (1995), S. Artavanis-Tsakonas et al., Science 268:225 (1995)). In nematodes, particular egg-laying defects associated with loss of sel-12 function are rescued by PS1 (D. Levitan et al., Proc. Natl. Acad. Sci., USA 93:14940 (1996)), indicating that for some functions, the homologous proteins might be functionally interchangeable.
PS1 has been linked in Alzheimer's Disease (AD), a neurological disorder that disproportionately affects the population over 65 years of age. Mutations in PS1 contribute to approximately 25% of early-onset familial Alzheimer's Disease. Incidence of the disease increases from less than 1% at age 60-65, to 5% at age 75, to as high as 47% at age 85. As a result, 60% to 80% of all cases of dementia in persons over age 65 are caused by AD. Afflicted individuals exhibit impaired cognitive function and memory.
Distinguishing features of AD include the presence of senile plaques as well as, neurofibrillary tangles and extensive neuronal loss in the neocortex, hippocampus and associated structures. The senile plaques are extracellular deposits of heterogeneous substances of which the major component is a 39-43 amino acid peptide referred to as &bgr;-amyloid peptide or A&bgr;(Glenner and Wong,Biochem. Biophys. Res. Commun. 120:885-890 (1984). The 4 kDa A&bgr; peptide is derived by proteolytic cleavage of a larger &bgr;-amyloid precursor protein (APP). The plaques are surrounded by a halo of dystrophic neurites, glia and astrocytes. &bgr;-amyloid deposits are also present in neocortex blood vessel walls. Other components of the plaques include ubiquitin, amyloid P, Apo E, interleukin-1, and a-i-antichymotrypsin. Although the complete etiology of AD has not yet been determined, much is now known, including genetic, immunological and environmental factors implicated in the development of AD.
Genetic data is seen from the study of familial AD. Although the majority of AD cases appear sporadic, about 10% of cases are early onset familial AD (FAD). Genetic analysis of FAD families has established that the disorder is associated with autosomal dominant inheritance of mutations in specific genes including the &bgr;-amyloid precursor protein (APP) located on chromosome 21, the PS1 gene on chromosome 14 and a homolog of PS1, presenilin 2 (PS2), located on chromosome 1. About 25% of the early-onset FAD cases are linked to mutations in PS1. Biochemical studies have shown that mutations in all three genes lead to an increased production of either the total A&bgr; or A&bgr;42(43), which is believed to be more amylodogenic (Scheuner et al., Nature Medicine 2:864-870 (1996); Selkoe, Science, 275:630-631 (1997)). To date, 42 different missense mutations and one in-frame splice site mutation were described (Cruts et al, Hum. Mol. Genet. 5:1449-1455 (1996)). However, neither all of the physiological roles of PS1, nor its mechanism in AD pathogenesis, are fully understood at this time.
Transgenic non-human animal technology offers a model system to address the effects of genes associated with AD. Addition of a gene construct directing the expression of human PS1 or its components to key regions in the central nervous system provide a means to study the expression and activity of PS1 and modulators of PS1. Previous attempts to express either the wild-type human PS1 protein or human PS1 containing various FAD mutations under either the PDGF promoter or the PrP promoter have been reported (Duff et al., Nature 383:710-713 (1996); Borchelt et al., Neuron 17:1005-1013 (1996)). In each case, the PS1 transgenes were reportedly expressed on the wild-type murine PS1 background. Thus, a mixture of murine and human PS1 proteins were produced in the resulting transgenic mice.
A relative increase of A&bgr;42(43) level has been observed in plasma and fibroblast cell cultures derived from PS1 FAD subjects and in transgenic mice expressing human PS1 FAD proteins (Scheuner et al, 1996; Duff et al., (1996); Borchelt et al, (1996); Lemere et al., (1996); Citron et al., Nature Medicine 3:67-72 (1997)). That result suggests that the pathogenic consequence of PS1 mutations might be through APP processing and A&bgr;42(43) production.
SUMMARY OF THE INVENTION
Accordingly, it is an aspect of this invention to provide a non-human transgenic animals which is heterozygous for a functional PS1 gene native to that animal. As used herein, functional is used to describe a gene or protein that, when present in a cell or in vitro system, performs normally as if in a native or unaltered condition or environment. The animal of this aspect of the invention is useful for the study of the tissue and temporal specific expression or activity of PS1 in an animal having only one functional copy of the gene. The animal is also useful for studying the ability of a variety of compounds to act as modulators of PS1 activity or expression in vivo or, by providing cells for culture, in vitro. As used herein, a modulator is a compound that causes a change in the expression or activity of PS1, or causes a change in the effect of the interaction of PS1 with its ligand(s), or other protein(s). In an embodiment of this aspect, the animal is used in a method for the preparation of a further animal which lacks a functional native PS1 gene. In another embodiment, the animal of this aspect is used in a method to prepare an animal which expresses a non-native PS1 gene in the absence of the expression of a native PS1 gene. In particular embodiments the non-human animal is a mouse. In further embodiments the non-native PS1 is a wild-type human PS1, an A246E mutant human PS1 gene or any other mutant human PS1 gene.
In reference to the transgenic animals of this invention, we refer to transgenes and genes. As used herein, a transgene is a genetic construct including a gene. The transgene is integrated into one or more chromosomes in the cells in an animal by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal. A gene is a nucleotide sequence that encodes a protein. The gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art.
Another aspect of the invention is a non-human animal embryo deficient for native PS1 expression. This embryo is useful in studying the effects of the lack of presenilin 1 on the developing animal. In particular embodiments the animal is a mouse. The animal embryo is also useful as a source of cells lacking a functional native PS1 gene. The cells are useful in in vitro culture studies in the absence of PS1.
An aspect of this invention is a method to obtain an animal in which the cells lack a functional gene PS1 native to the animal. The method includes providing a gene for an altered form of the PS1 gene native to the animal in the form of a transgene and targeting the transgene into a chromosome of the animal at the place of the native PS1 gene. The transgene can be
Jiang Ping
Qian Su
Sisodia Sangram S.
Van Der Ploeg Leonardus H. T.
Wong Philip Chun-Ying
Crouch Deborah
Merck & Co. , Inc.
Tribble Jack L.
Yablonsky Michael D.
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