Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-10-21
2001-03-27
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S018000, C800S003000
Reexamination Certificate
active
06207878
ABSTRACT:
BACKGROUND OF THE INVENTION
The dystrophin-glycoprotein complex (DGC) is a multi-subunit protein complex expressed at the sarcolemma of skeletal, cardiac, and smooth muscle fibers (reviewed in Campbell, Cell 80: 675-679 (1995), and Straub and Campbell, Curr. Opin. Neurol. 10: 169-175 (1997)). The DGC is currently known to be composed of at least nine proteins including dystrophin, the syntrophins, &agr;- and &bgr;-dystroglycan, &agr;-, &bgr;-, &ggr;-, and &dgr;-sarcoglycans and sarcospan. One of the functions of the DGC is likely to provide a structural link between the extracellular matrix and the actin cytoskeleton through interactions of dystrophin with filamentous actin, and &agr;-dystroglycan with the extracellular matrix component laminin, thereby maintaining the stability of the sarcolemma under contractile forces (Ervasti and Campbell,
J. Cell Biol
. 122(4): 809-823 (1993); and Campbell,
Cell
80: 675-679 (1995). Recent evidence suggests that the DGC may play other roles in normal muscle physiology through interactions with cell signaling molecules or other proteins at the sarcolemma.
Sarcospan is the most recently cloned component of the DGC (Crosbie et al.,
J. Biol. Chem
. 272: 31221-31224 (1997). Hydropathy plots predict that the protein has four transmembrane domains with an extracellular loop extending between transmembrane domains 3 and 4 (Scott et al., Genomics 20: 227-230 (1994); Crosbie et al.,
J. Biol. Chem
. 272: 31221-31224 (1997). Dendogram analysis designates sarcospan as a member of the tetraspan superfamily, also known as the transmembrane-4 superfamily or the tetraspanins (Heighway et al.,
Genomics
35: 227-230 (1996); Wright and Tomlinson,
Immunol. Today
15: 588-594 (1994). Tetraspan proteins are thought to function as molecular facilitators, mediating interaction between proteins at the plasma membrane. The tetraspans have also been implicated in cell adhesion, migration, and proliferation (Wright et al.,
Immunol. Today
15: 588-594 (1994); Maecher et al.,
FASEB
11: 428-442 (1997). Sarcospan is tightly associated with the sarcoglycans to form a subcomplex of the DGC (Crosbie et al.,
J. Cell Biol
. 145: 153-165 (1999). The function of the sarcoglycan-sarcospan complex is currently unknown. One hypothesis is that it stabilizes &agr;-dystroglycan at the membrane. Another hypothesis is that the sarcoglycan-sarcospan complex may be important in the signaling functions of the DGC, a possibility which remains relatively unexplored.
Defects in components of the DGC have been implicated in muscle disorders manifested by muscle weakness and wasting. Currently, it is known that six forms of muscular dystrophies are caused by primary genetic defects within components of the DGC. These include Duchenne and Becker muscular dystrophies, the most prevalent forms of muscular dystrophies that are caused by mutations in the dystrophin gene and four forms of autosomal recessive limb-girdle muscular dystrophies (LGMD2-C, -D, -E and -F) caused by primary mutations in each of the four sarcoglycan genes (Straub and Campbell,
Curr. Opin. Neurol
. 10: 169-175 (1997). Additionally, mutations in the laminin-&agr;2 chain cause a severe form of congenital muscular dystrophy. Recent data suggests that dystroglycan is important in basement membrane formation (Henry and Campbell,
Cell
95: 859-870 (1998) and dystroglycan-null mice die at a very early embryonic stage (Williamson et al.,
Hum. Mol. Genet
. 6: 831-841 (1997). It is likely that human-null mutations in the dystroglycans would also lead to an early embryonic lethality. In contrast, disruption of the &agr;1-syntrophin gene in mice was not lethal, and also did not result in muscle degeneration (Kameya et al.,
J. Biol. Chem
. 274: 2193-2200 (1999). However, neuronal nitric oxide synthase, which is usually localized at the sarcolemma through &agr;1-syntrophin, was not found at the sarcolemma in these animals (Kameya et al.,
J. Biol. Chem
. 274: 2193-2200 (1999).
To investigate the function of sarcospan, a sarcospan-deficient mouse was generated and characterized. The mouse generated was observed to exhibit an obese phenotype. Obesity in humans is a widespread and serious disorder, affecting a high percentage of the adult population in developed countries. Few persons suffering from this disorder are able to permanently achieve significant weight loss. This failure to treat obesity may be at least partially attributed to the complexity of the disease. An understanding of the genetic factors that underlie obesity may aid in treatment. Animal models are useful in developing this understanding. Current mouse models for obesity include obese (ob), agouti (wt), tubby (tub), fat and diabetes (db). These animal models are extremely useful for their ability to simplify the heritability of an otherwise very complex trait.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a mouse, and cells derived therefrom, homozygous for a disrupted sarcospan gene, wherein the disruption in the gene is introduced into the mouse or an ancestor of the mouse at an embryonic stage, wherein the disruption prevents the synthesis of functional sarcospan in cells of the mouse. The mouse is characterized as weighing substantially more, and having substantially larger deposits of white adipose tissue, as compared to an otherwise genetically identical mouse lacking a disrupted sarcospan gene. In one embodiment, the introduced disruption comprises a deletion in a portion of the sarcospan gene. One such deletion is a region of about 7.6 kb, including 223 base pairs of intron 1, the entire exon 2, the entire intron 2, and 1800 base pairs of exon 3, and replacement of the deleted region with a PGK-neomycin resistance cassette as a marker for neomycin resistance. This deletion can be made by introducing into embryonic stem cells, a DNA construct comprising 2132 base pairs of intron 1 and 2220 base pairs of exon 3 of the sarcospan gene; and a neomycin resistance gene inserted between the portion of intron 1 and exon 3 of the sarcospan gene listed in a), the neomycin resistance gene being in the opposite transcriptional orientation as the sarcospan exons replaced; wherein the introduced construct lacks 223 base pairs of intron 1, the entire exon 2, the entire intron 2, and 1880 base pairs of exon 3, of the sarcospan gene.
In another aspect, the present invention relates to a method for diagnosing an individual with a clinical disorder associated with reduced expression of sarcospan. The method comprises providing a tissue biopsy sample from the individual and then quantitatively detecting sarcospan expression in cells of the sample. Sarcospan expression is also quantitatively detected in a comparable control sample obtained from a control individual known to exhibit normal expression of sarcospan, by otherwise identical means. The amount of sarcospan expression detected in the sample is compared to the amount of sarcospan expression detected in the control sample, with a substantially lesser amount of sarcospan expression in the former by comparison being indicative of the clinical disorder in the individual. The tissue biopsy is obtained from either skeletal muscle, cardiac muscle, brown adipose tissue, white adipose tissue, smooth muscle, vascular smooth muscle. In one embodiment, detection of sarcospan expression is accomplished by immunoassay for the sarcospan protein. In another embodiment, detection of sarcospan expression is accomplished by detection of the sarcospan mRNA, preferably by RNA blot analysis or reverse transcriptase polymerase chain reaction.
In another aspect, the present invention relates to a method for diagnosing an individual with a clinical disorder associated with a mutation in the sarcospan gene. The method comprises isolating nucleic acids encoding the sarcospan gene, or a portion thereof, from the individual and then analyzing the nucleic acids by means to identify a mutation predicted to alter sarcospan expression or function in comparison to wild-type, with identification of the mutation being indi
Campbell Kevin P.
Crosbie Rachelle
Lebakken Connie
Williamson Roger
Farrell Kevin M.
Priebe Scott D.
Shukla Ram R.
University of Iowa Research Foundation
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