Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-03-16
2004-05-04
Falk, Anne-Marie (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S025000, C435S325000, C435S352000, C435S354000, C435S001100
Reexamination Certificate
active
06730821
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
Normal growth and differentiation of all organisms is dependent on cells responding correctly to a variety of internal and external signals. Many of these signals produce their effects by ultimately changing the transcription of specific genes. One well-studied group of proteins that mediate a cell's response to a variety of signals is the family of transcription factors known as nuclear receptors. Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis and trans retinoic acid, thyroid hormone, fatty acids (and other peroxisomal proliferators), as well as so-called orphan receptors, proteins that are structurally similar to other members of this group, but for which no ligands are known. Orphan receptors may be indicative of unknown signaling pathways in the cell or may be nuclear receptors that function without ligand activation. There are indications that the activation of transcription by some of these orphan receptors may occur in the absence of an exogenous ligand and/or through signal transduction pathways originating from the cell surface.
Steroid hormones affect the growth and function of specific cells by binding to intracellular receptors (SR) and forming SR-hormone complexes. SR-hormone complexes then interact with a hormone response element (HRE) in the control region of specific genes and alter specific gene expression. cDNAs for many SRs have been isolated and characterized, making it possible to deduce the amino acid sequences of various steroid/thyroid/retinoic acid receptors and related members of the super family of nuclear receptors (Evans et al.,
Science,
240:889-895 (1988); Liao et al.,
J. Steroid Biochem.,
34:(1-6) 41-51 (1989); Forman et al.,
New Biol.,
587-594 (1990)).
The complete coding sequences for human (AF148128) and murine (AF148129; SEQ ID NO:21) retina-specific nuclear receptors were determined and published by Chen et al. (
Proc. Natl. Acad. Sci. U.S.A.
96(26), 15149-15154 (1999)). According to Chen et al., human RNR is a splice variant of PNR. Northern blot and reverse transcription-PCR analyses of human mRNA samples demonstrated that RNR is expressed exclusively in the retina, with transcripts of approximately 7.5 kb, approximately 3.0 kb, and approximately 2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrated that RNR is expressed in the retinal pigment epithelium and in Muller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR).
Given the importance of retinoic acid receptors in the regulation of gene expression, a clear need exists for further characterization of these receptors 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. More specifically, the present invention relates to nucleic acid sequences encoding a retina-specific nuclear receptor and in the in vivo characterization of genes encoding a retina-specific nuclear receptor.
The present invention provides transgenic cells comprising a disruption in retina-specific nuclear receptor gene. Preferably, the transgenic cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells. Preferably, the target gene's coding sequence (i.e., exons) comprises SEQ ID NO:21. 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 disruption of the target sequence encoding a retina-specific nuclear receptor. In another embodiment, the transgenic cells are derived from the transgenic animals described below.
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 generating transgenic cells comprising a disruption in a retina-specific nuclear receptor. In one embodiment, the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the target sequence. The targeting construct also comprises a polynucleotide sequence that encodes a positive selection marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct.
The present invention further provides non-human transgenic animals comprising a disruption in a retina-specific nuclear receptor gene and methods of producing such transgenic animals. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the gene that naturally encodes and expresses a functional retina-specific nuclear receptor gene. In one aspect, the transgenic animals of the present invention are defective in the function of the retina-specific nuclear receptor gene. The present invention also encompasses cells and cell lines derived from the transgenic animals of the present invention.
The transgenic animals of the present invention further comprise a phenotype associated with having a defect or disruption in a retina-specific nuclear receptor gene.
The present invention also provides a method of identifying agents capable of affecting a phenotype of a transgenic animal. According to this method, a putative agent is administered to a transgenic animal. The response of the transgenic animal to the putative agent is then 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 further provides a method of identifying agents having an effect on retina-specific nuclear receptor gene expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse, having a disruption in a retina-specific nuclear receptor gene. 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 mouse. The response of the transgenic animal as compared to the control mouse may serve as an indication of the specificity or activity of the agent. Compounds that may have an effect on retina-specific nuclear receptor gene expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.
The present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption in a retina-specific nuclear receptor gene. In a preferred embodiment, conditions include those associated with the phenotypes of the mice of the present invention. In accordance with this method, the present invention provides animal models useful in identifying compounds that are able to affect a phenotype, such as a physiological or behavioral phenotype associated with a disruption of a retina-specific nuclear receptor gene. The method involves, for example, administering a putative agent to a transgenic animal. The response of the transgenic animal to the putative agent is then measured and compared to the response of a “normal” or wild-type mouse, or alternatively compared to a transgenic animal control (without agent administration)
Deltagen Inc.
Driscoll Robert J.
Falk Anne-Marie
Qian Celine
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