Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-07-06
2004-07-13
Falk, Anne-Marie (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S022000, C435S325000
Reexamination Certificate
active
06762342
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
Phosphatases represent unique and attractive targets for small-molecule inhibition and pharmacological intervention. Phosphatases comprise a widely varying group of enzymes that hydrolyze phosphomonoesters. Acid phosphatases are specific for the single-charged phosphate group and alkaline phosphatases for the double-charged group. More specifically, alkaline phosphatase (ALP) is an enzyme of the hydrolase class having a high (>8) pH optimum that catalyzes the cleavage of inorganic phosphate from phosphate esters. Alkaline phosphatase is important in recycling phosphate within living cells, and is found in high concentration in liver & biliary tract, bone, intestinal mucosa and placenta. Differing forms of the enzyme occur in normal and malignant tissues. The activity in serum is useful in the clinical diagnosis of many illnesses. Deficient bone enzyme activity causes hypophosphatasia.
There are three distinct isoforms of alkaline phosphatases: intestinal, placental, and liver/bone/kidney. The gene for intestinal alkaline phosphatase (abbreviated as IAP or ALPI) has been isolated and sequenced, and has been found to comprise 11 exons interrupted by 10 introns. Recently, a full-length human placental alkaline phosphatase (AP) cDNA was used to identify and clone related genes from mouse genomic libraries (
Genomics
8(3), 541-54 (1990)). The deduced amino acid sequence of the isozymes predicts proproteins of 529, 559, and 466 amino acids for embryonic AP, intestinal AP, and pseudo-AP, respectively. A repetitive sequence inserted in exon XI of the mouse intestinal AP gene codes for a unique stretch of 41 amino acids, 20 of which are threonines. This insertion has disrupted a region recognized as being responsible for phosphatidylinositol anchorage of human placental AP to the cytoplasmic membrane.
Given the importance of phosphatases, and alkaline phosphatases especially, a clear need exists for identification and characterization of phosphatases 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 an intestinal alkaline phosphatase 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 intestinal alkaline phosphatase 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 intestinal alkaline phosphatase 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 an intestinal alkaline phosphatase gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the intestinal alkaline phosphatase gene. In one aspect, the transgenic animals of the present invention are defective in the function of the intestinal alkaline phosphatase gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in an intestinal alkaline phosphatase gene. In a preferred embodiment, the non-human transgenic animals of the present invention demonstrate a nociceptive disorder. In another preferred embodiment, the non-human transgenic animals of the present invention demonstrate increase in thermal sensitivity. In another preferred embodiment, the non-human transgenic animals of the present invention demonstrate an increased pain response. In yet other preferred embodiments, the non-human transgenic animals of the present invention demonstrate hypoactivity or reduced anxiety.
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 intestinal alkaline phosphatase gene.
The present invention further provides a method of identifying agents having an effect on intestinal alkaline phosphatase 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 intestinal alkaline phosphatase 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 an intestinal alkaline phosphatase gene. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the intestinal alkaline phosphatase gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the intestinal alkaline phosphatase gene, comprising the steps of contacting the intestinal alkaline phosphatase gene with an agent and detecting an agent/intestinal alkaline phosphatase gene complex. Such complexes can be detected by, for example, measuring expression of an operably linked detectable marker.
The invention further provides methods of treating diseases or conditions associated with a disruption in an intestinal alkaline phosphatase gene, and more particularly, to a disruption in the expression or function of the intestinal alkaline phosphatase gene. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a disruption in the intestinal alkaline phosphatase gene's expression or function, including administering to a subject in need, a therapeutic agent that effects intestinal alkaline phosphatase expression or function. In accordance with this embodiment, the method comprises administration of a therapeutically effective amount of a natural, synthetic, semi-synthetic, or recombinant intestinal alkaline phosphatase gene, intest
Deltagen Inc.
Driscoll Robert J.
Falk Anne-Marie
Qian Celine
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