Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-06-29
2004-08-31
Falk, Anne-Marie (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S021000, C800S025000, C435S325000
Reexamination Certificate
active
06784335
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function.
BACKGROUND OF THE INVENTION
Generally speaking, growth factors are proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation. Many growth factors are quite versatile, stimulating cellular division in numerous different cell types; while others are specific to a particular cell-type. More specifically, growth factors are substances, such as polypeptide hormones, which affect the growth of defined populations of animal cells in vivo or in vitro, but which are not nutrient substances. Proteins involved in the growth and differentiation of tissues may promote or inhibit growth, and promote or inhibit differentiation, and thus the general term “growth factor” includes cytokines, trophic factors, and their inhibitors. Among growth, or neurotrophic, factors presently known are the transforming growth factors (TGF-alpha, TGF-beta, TGF-gamma). Transforming growth factor-beta appears to elicit a variety of responses in many different cell types.
Widespread neuronal cell death accompanies normal development of the central and peripheral nervous systems. Studies of peripheral target tissues during development have shown that neuronal cell death results from the competition among neurons for limiting amounts of survivor factors (“neurotrophic factors”). The earliest identified of these, nerve growth factor (“NGF”), is the most fully characterized and has been shown to be essential for the survival of sympathetic and neural crest-derived sensory neurons during early development of both chick and rat. Barde et al., U.S. Pat. No. 5,229,500, describe nucleic acid sequences encoding brain derived neurotrophic factor (“BDNF”), as well as the BDNF protein. BDNF is suggested for treating Parkinson's Disease and Alzheimer's Disease. Additional uses are for the identification of homologous regions between BDNF and NGF so as to identify and isolate additional members of the NGF family, and also to generate immunogen by antibodies directed toward BDNF or fragments.
Among TGF-beta members are the bone morphogenetic proteins (BMP). The BMPs have been indicated as useful in wound healing, tissue repair, and to induce cartilage and/or bone growth. BMPs have potent effects during embryogenesis. One member, BMP-4, has been shown to have potent ventralizing effects in Xenopus embryos, leading to the differentiation of blood and mesenchyme and inhibiting the formation of dorsal tissues such as notochord, muscle, and nervous system (see, e.g., Jones et al.,
Development
115:639-647 (1991)). BMP-4 is expressed ventrally in the Xenopus embryo and its expression is increased by ventralizing treatments such as irradiation with ultraviolet light.
During animal development, cells exchange signals with their neighbors that allow specific groups of cells to produce organized, differentiated structures such as limbs, segments or other body parts. Understanding these processes might allow one to manipulate them to regenerate damaged body parts or to diagnose birth defects. The twisted gastrulation protein (TSG) is one of five known secreted proteins required to pattern the dorsal part of the early Drosophila embryo. Unlike the decapentaplegic (DPP) protein that is required to pattern the entire dorsal half of the embryo, TSG is needed only to specify the fate of the dorsal midline cells. When expressed in a ventral stripe of cells, TSG protein can diffuse to the dorsal-most cells and can rescue the dorsal midline cells in tsg mutant embryos. TSG functions in a permissive rather than instructive role to elaborate cell fates along the dorsal midline after peak levels of DPP activity have ‘primed’ cells to respond to TSG.
Given the importance of growth factors, a clear need exists for identification and characterization of growth factors which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
SUMMARY OF THE INVENTION
The present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function.
The present invention provides transgenic cells comprising a disruption in a BMP gene. The transgenic cells of the present invention are comprised of any cells capable of undergoing homologous recombination. Preferably, the cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells. According to one embodiment, the transgenic cells are produced by introducing a targeting construct into a stem cell to produce a homologous recombinant, resulting in a mutation of the BMP gene. In another embodiment, the transgenic cells are derived from the transgenic animals described below. The cells derived from the transgenic animals includes cells that are isolated or present in a tissue or organ, and any cell lines or any progeny thereof.
The present invention also provides a targeting construct and methods of producing the targeting construct that when introduced into stem cells produces a homologous recombinant. In one embodiment, the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the BMP gene. The targeting construct also comprises a polynucleotide sequence that encodes a selectable marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct. The targeting construct may also comprise other regulatory elements that may enhance homologous recombination.
The present invention further provides non-human transgenic animals and methods of producing such non-human transgenic animals comprising a disruption in a BMP gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the BMP gene. In one aspect, the transgenic animals of the present invention are defective in the function of the BMP gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a BMP gene. In a preferred embodiment, the non-human transgenic animals of the present invention comprise abnormalities in nociception. In another preferred embodiment, the non-human transgenic animals of the present invention demonstrate a decrease in response to pain. In yet another embodiment, the non-human transgenic animals of the present invention demonstrate an anxiety disorder.
The present invention also provides methods of identifying agents capable of affecting a phenotype of a transgenic animal. For example, a putative agent is administered to the transgenic animal and a response of the transgenic animal to the putative agent is measured and compared to the response of a “normal” or wild type mouse, or alternatively compared to a transgenic animal control (without agent administration). The invention further provides agents identified according to such methods. The present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption of the BMP gene.
The present invention further provides a method of identifying agents having an effect on BMP expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse. The method includes measuring a response of the transgenic animal, for example, to the agent, and comparing the response of the transgenic animal to a control animal, which may be, for example, a wild-type animal or alternatively, a transgenic animal control. Compounds that may have an effect on BMP expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.
The invention also provides cell lines comprising nucleic acid sequences of a BMP gene. Such cell lines may be capable of expressing such sequences by
Deltagen Inc.
Deltagen, Inc.
Falk Anne-Marie
Qian Celine
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