Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-05-05
2001-05-29
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S008000, C800S009000, C800S025000, C435S354000, C435S455000, C435S029000
Reexamination Certificate
active
06239326
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to genetically altered non-human animals, and particularly, to knockout animals, i.e., animals lacking a native gene function.
BACKGROUND OF THE INVENTION
SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin and BM-40, is an extracellular matrix (ECM)-associated glycoprotein widely distributed in human and murine tissues undergoing developmental regulation. While its specific role remains elusive, the high degree of interspecies conservation suggests a strong evolutionary pressure to conserve this protein. Extensive studies thus far have implicated SPARC in diverse functions including cell proliferation [Lane and Sage,
FASEB J
., 8:163-173 (1994)].
ECM proteins are required for growth factor stimulated cell proliferation since anchorage dependent cells grown as suspension cultures in the absence of ECM die eventually. However, the role of individual ECM proteins to support the growth induced by individual growth factor is not known. SPARC has been shown to influence cell growth in a cell type-specific manner. Down-regulation of SPARC did not perturb melanoma cell growth, while over-expression in ovary cancer cell lines or addition of purified SPARC to endothelial cells retarded cell growth [Ledda et al,
Nature Med
., 3:171-176, (1997); Mok et al.,
Oncogene
, 12:1895-1901 (1996); Funk and Sage,
J. Cell. Phsiol
., 154:53-63 (1993)]. Nevertheless, peptides corresponding to SPARC domain II stimulate cell proliferation [Funk and Sage,
J. Cell. Physiol
., 154:53-63 (1993)]. This result strongly suggests that extracellular cleavage may have important consequences on the ability of SPARC to modulate cell growth. Furthermore, SPARC was shown to interact with growth factors, cytokines and ECM components [Lane and Sage,
FASEB J
., 8:163-173 (1994)], indicating that SPARC may profoundly influence cell growth through these diverse functions.
The role of SPARC in cell proliferation is further supported by its expression in sprouting endothelial cells and endothelial cords during angiogenesis and in fibroblasts and macrophages migrating into wound sites [Sage and Bornstein,
J. Biol. Chem
., 266:14831-14834 (1991); Reed et al.,
J. Histochem. Cytochem
., 41:1467-1477 (1993)]. Moreover, SPARC is highly expressed in actively dividing osteoblasts, hypertrophic chondrocytes at growth plates and in developing embryos [Lane and Sage, cited above].
SPARC is abundantly expressed in extraembryonic parietal endoderm [Mason et al,
EMBO J
., 5:1465 (1986)], which specializes in the synthesis and secretion of a thick basement membrane known as Reichert's membrane or the parietal yolk sac. This membrane acts as a barrier keeping embryonic fluid inside to provide nutrients to embryos and to protect embryos from outside shock. SPARC is expressed in the heart, lung and somites of embryos. Somites differentiate into vertebrae, ribs, muscles and dermis of skin. Transgenic nematodes over-expressing SPARC are abnormal. Embryos are deformed, and adults show reduced mobility and paralysis, suggesting the involvement of SPARC in the adhesion of muscle cells to the ECM [Schwarzbauer and Spencer,
Mol. Biol. Chem
., 4:941-952 (1993)]. Microinjection of SPARC antibodies into Xenopus embryos resulted in abnormal axial development and developmental arrest at the neurula and tailbud stages [Purcell et al.,
J. Exp. Zool
., 265:153-164 (1993)].
There is a need in the art, especially in the medical and pharmaceutical fields, for commercial research and diagnostic systems useful for testing cell proliferation and screening for diseases resulting from impaired cell proliferative processes.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides a fertile, transgenic non-human mammal having a phenotype characterized by the substantial absence of SPARC. This mammal is preferably a mouse. The transgenic animals of the invention provides a system useful for screening age-related conditions such as impaired wound healing, cataracts, diabetes mellitus, nephropathy and osteoporosis.
In another aspect, the invention provides a method for producing a transgenic non-human mammal having a phenotype characterized by the substantial absence of SPARC otherwise naturally occurring in the animal. This method involves introducing a transgene into an embryonic stem cell which disrupts the function of the native SPARC gene; injecting the cell into a blastocyst; transplanting the blastocyst into a pseudopregnant mammal; allowing the embryo to develop to term; identifying at least one transgenic offspring containing the transgene; and breeding said offspring to form a transgenic SPARC deficient animal having the phenotype.
In yet another aspect, the present invention provides a recombinant mammalian cell containing a transgene which causes a disruption in the function of SPARC.
In still another aspect, the present invention provides a method for screening drugs useful for the treatment of impaired wound healing, cataracts, diabetes mellitus, nephropathy and osteoporosis by utilizing the SPARC-deficient transgenic non-human mammals of the invention or cell cultures derived therefrom.
In yet another aspect, the present invention provides compositions and methods for modulating SPARC expression. These methods include use of polynucleotide molecules, such as viral and plasmid vectors, containing SPARC polynucleotide sequences.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
REFERENCES:
patent: WO 98/20112 (1998-05-01), None
J. Schwarzbauer and C. Spencer, “TheCaenorhabditis elegansHomologue of the Extracellular Calcium Binding Protein SPARC/Osteonectin Affects Nematode Body Morphology and Mobility,”Mol. Biol. Chem., 4:941-952 (Sep. 1993).
L. Purcell et al., “Developmental Anomalies of Xenopus Embryos Following Microinjection of SPARC Antibodies,”J. Exp. Zool., 265:13-164 (Feb. 1, 1993).
M. Evans et al., “Establishment in Culture of Pluripotential Cells fro Mouse Embryos,”Nature, 292:154-156 (Jul. 1981).
A. Poustka et al., “Selective Isolation of Cosmid Clones by Homologous Recombination inEscherichia coli,”Proc. Natl. Acad. Sci. USA, 81:4129-4133 (Jul. 1984).
C. C. Howe et al., “Expression of SPARC/osteonectin Transcript in Murine Embryos and Gonads,”Differentiation, 37:20-25 (1988).
J.H. McVey et al., “Characterization of the Mouse SPARC/Osteonectin Gene,”J. Biol. Chem., 263(23)11111-11116 (Aug. 15, 1988).
S. Thompson et al., “Germ Line Transmission and Expression of a Corrected HPRT Gene Produced by Gene Targeting in Embryonic Stem Cells,”Cell, 56:313-321 (Jan. 27, 1989).
Bradley et al (1992) Bio/Technology 10, 534-539.*
Mullins et al (1996) J. Clin. Invest. 98. S37-S40.*
Seamark et al (1994) Reprod. Fertil. Dev. 6, 653-657.*
Moreadith et al (1997) J. Mol. Med. 75, 208-216.*
Capecchi (Mar. 1994) Scientific American, 34-41.*
T. Lane and E. Sage, “The Biology of SPARC, a Protein That Modulates Cell-Matrix Interactions,”FASEB J., 8:163-173 (Feb. 1994).
M. Ledda et al. “Suppression of SPARC Expression by Antisense RNA Abrogates the Tumorigenicity of Human Melanoma Cells,”Nature Med., 3(2):171-176 (Feb. 1997).
S. Mok et al., “SPARC, an Extracellular Matrix Protein with Tumor-Suppressing Activity in Human Ovarian Epithelial Cells,”Oncogene, 12:1895-1901 (May 2, 1996).
S. Funk and E. Sage, “Differential Effects of SPARC and Cationic SPARC Peptides on DNA Synthesis by Endothelial Cells and Fibroblasts,”J. Cell. Physiol., 154:53-63 (Jan. 1993).
E. Sage and P. Bornstein, “Extracellular Proteins that Modulate Cell-Matri Interactions,”J. Biol. Chem., 266(23):14831-14834 (Aug. 15, 1991).
M. Reed et al., “Differential Expression of SPARC and Thrombospondin 1 in Wound Repair: Immunolocalization and In Situ Hybridization ,”J. Histochem. Cytochem., 41(10):1467-1477 (Oct. 1993).
I. Mason et al., “Evidence from Molecular Cloning that SPARC, a Major Product of Mouse Emb
Crouch Deborah
Howson and Howson
The Wistar Institute of Anatomy and Biology
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