Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-07-08
2002-12-31
Wilson, Michael C. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000
Reexamination Certificate
active
06501003
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to a transgenic mouse expressing green fluorescent protein under the control of a human glial fibrillary protein promoter and to a method of determining the neurotoxicity of substances in vivo.
BACKGROUND OF THE INVENTION
Neural tissue consists of neurons and supporting or glial cells. Glial cells outnumber neurons by about ten to one in the mammalian brain. Glial cells may be divided into four classes: astrocytes, oligodendrocytes, ependymal cells and microligal cells. Astrocytes descend from a primitive neuroepithelial stem cell line within the ependymal zone. The exact function of astrocytes is unknown. Astrocytes probably provide support for the delicate neurons, contribute to the synthesis and degradation of neurotransmitters, control the ionic environment of the neurons and provide spacing between neurons.
Glial fibrillary acidic protein (GFAP) is expressed primarily in astrocytes of the central nervous system (including Mueller cells of the retina and non-myelinating Schwann cells of the peripheral nervous system). GFAP is a 50 kD intracytoplasmic protein that is the primary intermediate filament in the cytoskeleton of astrocytes. Mouse and human GFAP genomic genes have been cloned and sequenced as described in U.S. Pat. No. 5,267,047, incorporated herein by reference. The GFAP gene contains a basal promoter consisting of a TATA box and a CAAT box. Several enhancer and silencer sequences have also been identified. The enhancers for GFAP expression are found between −250 and −80 bp and between −1980 and −1500 bp. These positive control regions contain consensus sequences for many transcription factors including a cAMP response element and binding sites for the Sp-1, NF-1, AP-1 and AP-2 transcription factors. Tissue specificity is conferred by a human GFAP consensus sequence (hgcs) located in the −1980 to −1500 bp region. The transactivating protein which binds to this site has not been identified.
Reactive gliosis (also known as astrogliosis) occurs in response to almost any insult, physical or chemical, to the central nervous system (CNS). Reactive gliosis is characterized by hypertrophy of the astrocyte cell body and its processes, accompanied by an increase in expression of GFAP. One of the major problems in neurotoxicity screening is the diversity of insults that are to be tested and the highly specific nature of their targets, particularly for the pharmacological agents that may affect very discrete populations of neurons. Reactive gliosis in glial cells, in which up-regulation of GFAP is an invariant part, represents a robust change in the central nervous system following injuries to all of the relevant cell types in the central nervous system (neurons, oligodendrocytes, vascular elements, and astrocytes themselves). In the peripheral nervous system, a similar increase in GFAP occurs following both traumatic and toxic injuries tp peripheral nerve [(Mancardi et al.,
J.Neurosci
. 102, 177 (1991); Toews et al.,
J.Neurosci
. 12, 3676 (1992); Quattrini et al.,
Glia
17, 294 (1996)]. In frogs, peri-synaptic Schwann cells at the neuromuscular junction also respond to degeneration of the nerve terminals by forming sprouts and increasing expression of GFAP.
The correlation between the upregulation of GFAP expression and neural injury has been recognized as providing a possible biochemical indicator of neurotoxic or physical damage to the CNS. (See Mucke,
The New Biologist
, Vol. 3, No. 5, 465 (May 1991); O'Callaghan,
Neurotoxicology and Teratology
, Vol. 13, 275 (1991); O'Callaghan,
Psychopharmacology Bulletin
30, 549 (1994); Verderber et al.,
Invest. Ophthalmol. Visual Sci
. 36, 1137 (1995); and Wu et al.,
J.Neurosci. Res
. 51, 675 (1998).) Mice transgenic for a GFAP-lacZ transgene exhibit increased production of the fusion protein in astrocytes of physically damaged brain and retina neural tissue. Likewise, exposure of mice to chemical neurotoxins results in increased wild-type GFAP expression as measured by immunohistochemistry and ELISA assays. Reactive gliosis in response to neurotoxin challenge is dose-, time-, and region-dependent. ELISA assays indicate that gliosis occurs at toxicant levels below those that cause light microscopic evidence of cell loss or damage.
The green fluorescent protein (GFP), a single peptide of 238 amino acids derived from the jellyfish
Aequorea victoria
, absorbs blue light and emits green light without a requirement for any cofactor or substrate. After the formation of its fluorophore by endogenous posttranslational cyclization, GFP is quite stable and remains fluorescent even after the harsh treatments found in many biochemical assays, such as 1% sodium dodecyl sulphate (SDS), 4% formaldehyde, and incubation at 65° C. Since the first report of its use in
Escherichia coli
and
Caenorhabditis elegans
by Chalfie et al.,
Science
263, 802 (1994), GFP has found many applications as a reporter gene in a number of higher organisms including Drosophila [Wang et al.,
Nature
369, 400 (1994)] and zebrafish [Amsterdam et al.,
Dev. Biol
. 171, 123 (1995); Peters et al.,
Dev. Biol
. 171, 252 (1995)].
The versatility of the GFP is enhanced by its ability to remain fluorescent as a fusion protein allowing studies of the subcellular distribution and dynamics of various proteins, including NMDA receptors [Marshall et al.,
Neuron
14, 211 (1995); Niswender et al.,
J. Microsc
. 180, 109 (1995); Aoki et al.,
FEBS Lett
. 384, 193 (1996)]. Recently, a “humanized” version of GFP has become available in which silent mutations were introduced to alter the codons to those more commonly used in mammals. The “humanized” GFP is generally expressed at higher levels in mammalian systems than wild-type GFP. Mutant forms of GFP have become available which emit light of greater intensity or which exhibit wavelength shifts. (See Clontech Catalogue, 1998).
These genetically altered proteins offer increased sensitivity in assays for measuring neural insult. They offer an opportunity to assess the toxicity of substances at much lower levels than heretofore possible with conventional approaches. For example, WO94/17208 discloses a method of assessing toxicity by independent measurement of the expression of four different types of stress promoters. Detection of increased levels of stress gene expression is effected either by nucleic acid hybridization or a reporter gene such as the genes encoding glutathione transferase, luciferase, chloramphenicol acetyl transferase, or galactose kinase.
Another conventional approach is the use of cell cultures in studying gliosis, as reviewed recently by Wu, et al., supra. Astrocytes were cultured or co-cultured with other cell types under a variety of conditions to establish a baseline under one or more biochemical or morphological parameters, and then the baselines compared to cells subjected to various damaging sources of stress. Glial markers include GFAP, vimentin and trophic factor.
SUMMARY OF THE INVENTION
Over the past several years considerable effort has gone into the development of non-invasive imaging techniques for studies of tissue structure, metabolism, and most recently, gene expression. Non-invasive imaging in neurotoxicity screening would offer particular benefits in that testing would not require sacrifice of the animal, thereby reducing costs and improving animal welfare. Scientifically, a major advantage would be the possibility of repeat measurements on the same animal over time, to assess longer term effects of potentially toxic substances.
Accordingly, it is an object of the present invention to provide an assay system free of the artifacts of tissue culture. It is also an object of the present invention to provide assays for physical and neurotoxic challenges to the nervous system, more sensitive to low dose toxicants than conventional methods. It is a still further object to provide a non-invasive assay of neurologic toxicity capable of monitoring toxic eff
Messing Albee
Zhuo Lang
Welch Teresa J.
Wilson Michael C.
Wisconsin Alumni Research Foundation
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