Genomic DNA fragments containing regulatory and coding...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S320100, C435S325000, C435S235100, C530S350000, C536S024100, C536S023100, C536S023500

Reexamination Certificate

active

06177242

ABSTRACT:

BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to DNA and clones of &bgr;2-subunit of neuronal nicotinic acetylcholine receptor (nAChR) sequences. This invention also relates to genomic DNA fragments containing regulatory and coding sequences for the &bgr;2-subunit neuronal nAChR and transgenic animals made using these fragments or mutated fragments. The 5′ flanking sequences contain a promoter, which confers neuron-specific expression. The genomic clones demonstrate the importance of the &bgr;2-subunit gene in the nicotinic system and in the pharmacological response to nicotine. The invention also relates to vectors containing the DNA sequences, cells transformed with the vectors, transgenic animals carrying the sequences, and cell lines derived from these transgenic animals. In addition, the invention describes the uses of all of the above.
References cited in this specification appear at the end by author and publication year or by cite number.
Neuron-specific expression. Many recombinant DNA-based procedures require tissue-specific expression. Unwanted or potentially harmful side-effects of gene transfer therapies and procedures can be reduced through correct tissue-specific expression. Furthermore, the ability to direct the expression of certain proteins to one cell type alone advances the ability of scientists to map, identify or purify these cells for important therapeutic or analytical purposes. Where the cells of interest are neurons or a particular subset of neurons, a need for DNA sequences conferring neuron-specific or subset-specific expression exists.
Proteins expressed throughout an organism are often utilized for specific purposes by neurons. By expressing a particular subunit or component of these proteins solely in neuronal tissue, the neuron tailors the protein activity for its purposes. Finding the particular, neuron-specific subunits or components and unraveling why they are produced only in neuronal tissue holds the key to DNA elements conferring neuron-specific expression. The inventors' knowledge of the biology of acetylcholine receptors provided an important foundation for this invention (see Changeux, The New Biologist, vol. 3, no.5, pp. 413-429). Different types of acetylcholine receptors are found in different tissues and respond to different agonists. One type, the nicotinic acetylcholine receptor (nAChR), responds to nicotine. A subgroup of that type is found only in neurons and is called the neuronal nAChR.
Generally, five subunits make up an acetylcholine receptor complex. The type of subunits in the receptor determines the specificity to agonists. It is the expression pattern of these subunits that controls the localization of particular acetylcholine receptor types to certain cell groups. The genetic mechanisms involved in the acquisition of these specific expression patterns could lead to an ability to control tissue-specific or even a more defined cell group-specific expression. The inventors' work indicates that defined elements in the promoter sequence confer neuron specific expression for the &bgr;2-subunit.
The Pharmacological Effects of Nicotine. As noted above, nAChR responds to the agonist nicotine. Nicotine has been implicated in many aspects of behavior including learning and memory (1,2). The pharmacological and behavioral effects of nicotine involve the neuronal nAChRs. Studies using low doses of nicotine (23) or nicotinic agonists (16) suggest that high affinity nAChRs in the brain mediate the effects of nicotine on passive avoidance behavior. Model systems where neuronal nAChR has been altered can therefore provide useful information on the pharmacological effects of nicotine, the role of neuronal nAChR in cognitive processes, nicotine addiction, and dementias involving deficits in the nicotinic system.
Functional neuronal nAChRs are pentameric protein complexes containing at least one type of &agr;-subunit and one type of &bgr;-subunit (3-5) (although the &agr;7-subunit can form functional homooligomers in vitro
6,7
). The &bgr;2-subunit was selected for this study from among the 7 known &agr;-subunits and 3 known &bgr;-subunits (3) because of its wide expression in the brain (8-10), and the absence of expression of other &bgr;-subunits in most brain regions (10). Mutation of this subunit should therefore result in significant deficits in the CNS nicotinic system. The inventors have examined the involvement of the &bgr;2-subunit in pharmacology and behavior. Gene targeting was used to mutate the &bgr;2-subunit in transgenic mice.
The inventors found that high affinity binding sites for nicotine are absent from the brains of mice homozygous for the &bgr;2-subunit mutation, &bgr;2−/−. Further, electrophysiological recording from brain slices reveals that thalamic neurons from these mice do not respond to nicotine application. Finally, behavioral tests demonstrate that nicotine no longer augments the performance of &bgr;2−/−mice on the test of passive avoidance, a measure of associative learning. Paradoxically, mutant mice are able to perform better than their non-mutant siblings on this task.
BRIEF SUMMARY OF THE INVENTION AND ITS UTILITY
In an aspect of this invention, we describe a 15 kb fragment of DNA carrying regulatory and coding regions for the &bgr;2-subunit of the neuronal nAchR. We characterize the promoter of the &bgr;2-subunit gene in vitro and in transgenic mice. We describe several DNA elements, including an E-box and other consensus protein-binding sequences involved in the positive regulation of this gene. Moreover, we show that the cell-specific transcription of the &bgr;2-subunit promoter involves at least two negative regulatory elements including one located in the transcribed sequence.
Preferred embodiments of these aspects relate to specific promoter sequences and their use in directing neuron-specific expression in various cells and organisms. An 1163 bp sequence and an 862 bp sequence both confer neuron-specific expression. Other embodiments include the −245 to −95 sequence of
FIG. 1
, containing an essential activator element, and the −245 to −824 sequence of
FIG. 1
containing a repressor. A repressor element composed of the NRSE/RE1 sequence is also present in the transcribed region. Certain plasmids comprising these genomic sequences are described as well.
The promoter sequences are important for their ability to direct protein, polypeptide or peptide expression in certain defined cells. For example, in the transgenic mice as shown below, proteins encoding toxins or the like can be directed to neurons to mimic the degradation of those cells in disease states. Others will be evident from the data described below.
Alternatively, the promoters can direct encoded growth factors or oncogenic, tumorigenic, or immortalizing proteins to certain neurons to mimic tumorigenesis. These cells can then be isolated and grown in culture. In another use, the promoter sequences can be operatively linked to reporter sequences in order to identify specific neurons in situ or isolate neurons through cell sorting techniques. The isolated, purified neurons can then be used for in vitro biochemical or genetic analysis. Reporter sequences such as LacZ and Luciferase are described below.
In another aspect of this invention, the inventors provide the genomic clones for mouse &bgr;-2 subunit of the neuronal nAChR. These clones are useful in the analysis of the mammalian nicotinic system and the pharmacology of nicotine. The inventors describe assays using transgenic mice where the genomic clones of the &bgr;2-subunit have been used to knock out the high affinity binding of nicotine.
In addition to the deletion mutants described, mutations incorporated into the exons or regulatory sequences for the &bgr;2-subunit will result in useful mutant transgenic animals. These mutations can be point mutations, deletions or insertions that result in non-efficient activity of the nAChR or even a non-active receptor. With such mutant animals, methods

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