Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1999-10-27
2001-11-13
Clark, Deborah J. R. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S003000, C800S009000, C800S014000, C800S025000, C424S009200, C435S325000, C435S353000
Reexamination Certificate
active
06316689
ABSTRACT:
TECHNICAL FIELD
This invention relates to 25-hydroxyvitamin D
3
24-hydroxylase transgenic animals.
BACKGROUND ART
The transgenic animal carries a gene introduced to its own or progenitor's germ cell line in an early stage of development (usually, single cells).
Wagner et al. (1981, ProNAS, USA, 78, 50169 and Stewart et al. (1982, Science, 217, 1046) describe transgenic mice carrying the human globin gene. Constantini et al. (1981, Nature, 294, 92) and Lacy et al. (1983, Cell, 34, 343) describe transgenic mice carrying the rabbit globin gene. McKnight et al. (1983, Cell, 34, 335) describe mice carrying a foreign transferrin gene. Brinstar et al. (1983, Nature, 306, 332) describe mice carrying a functionally introduced immunoglobulin gene.
With regard to transgenic mice carrying a foreign gene related to diseases of bone, there is the following report. Lewis, D. B. et al. (1993, ProNAS, USA, 90, 11618) describe mice carrying the mouse lck-IL4 gene introduced and report that the osteocalcin level in those mice was significantly low, with the animals manifesting osteoporosis-like symptoms.
Meanwhile, as mice carrying a foreign gene related to diseases of the kidney, the following reports are available. Doi, T. et al. (1988, Am. J. Pathol., 131, 398) describe mice carrying the bovine growth hormone gene introduced and report that those mice showed diffuse mesangial hyalinization at week 7 of age and advanced glomerulosclerosis at week 30 or later, and subsequently died of uremia.
Dressler, G. R. et al. (1993, Nature, 362, 65) describe mice carrying the Pax-2 gene introduced and report that, in their kidneys, expression of the gene could be confirmed and nephrotic symptoms such as glomerular atrophy and proteinuria were observed. Lowden, D. A. et al. (1993, J. Lab. Clin. Med., 124, 386) describe mice carrying a foreign TGF-&agr; gene introduced and report that expression of the gene in the kidneys could be confirmed and such symptoms as cyst formation and glomerular hypertrophy were observed.
However, there is not known a rat carrying any foreign gene introduced which can serve as a model of bone disease or kidney disease.
It is known that vitamin D exists in the natural kingdom as two native forms, D
2
and D
3
, and that whereas D
2
has a double bond at the 22-position and a methyl group at the 24-position of its side chain and occurs in plants, D
3
occurs in animals. As the 7-hydrocholesterol (provitamin D
3
) produced in the human skin is exposed to ultraviolet light from 290 to 320 nm, it undergoes photodegradation involving cleavage of the B ring at the 9, 10-position to give provitamin D
3
. This, in turn, is isomerized at body temperature to give vitamin D
3
. The structure of vitamin D
3
contains 3 double bonds, resulting from cleavage of the B ring, in every other positions. Vitamin D
3
is coupled to a vitamin D-binding protein and the couple is transported to the liver, where the 25-position of its side chain is hydroxylated by the enzyme 25-hydroxylase present in the liver cell mitochondria to give 25-hydroxyvitamin D
3
. This substance is thence transported to the kidneys and, depending on the calcium metabolism regulating hormones in the bloodstream, such as PTH, and the blood calcium concentration, it is further hydroxylated at the 1&agr;-, 23-, 24- and 26-positions in the proximal renal tubules to give 1&agr;,25-dihydroxyvitamin D
3
, 23,25-dihydroxyvitamin D
3
, 24,25-dihydroxyvitamin D
3
and 26,25-dihydroxyvitamin D
3
, respectively. Among them, the metabolite having high biological activity is 1&agr;,25-dihydroxyvitamin D
3
, which elevates blood calcium and phosphorus levels and discharges bone metabolizing functions; namely it assists in the expression of osteopontin and osteocalcin by osteoblasts, inhibits production of proteoglycan and, conversely, enhances production of cell membrane-derived phospholipids to thereby accelerate calcification of the osteoblasts. Regarding the function of 24,25-dihydroxyvitamin D
3
, this substance is known to inhibit formation of osteoclasts, promote expression of the osteocalcin gene and further reportedly accelerate calcification in vitamin D-deficient rats but this action is dependent on the 25-hydroxyvitamin D
3
24-hydroxylase (also called vitamin D
3
24-hydroxylase or 1&agr;,25-dihydroxyvitamin D
3
-hydroxylase) which is present in the proximal renal tubules. Furthermore, recent studies have revealed that this enzyme hydroxylates not only the 24-position but also the 26-position.
Among important known disorders of vitamin D metabolism are vitamin D-dependent disease Type II, rickets, osteomalacia, and so forth. Those diseases manifest dysosteogenesis and deformity of bone resulting from a disorder of calcification, and the occurrence of this disorder prior to closure of the epiphyses leads to rickets, while occurrence of the disorder at later times leads to osteomalacia. Aside from the above diseases, there exist risks for renal diseases inclusive of renal insufficiency, secondary hyperparathyroidism and hypercalcemia occurring either as complications of said diseases or independently.
Ohyama et al. (1989, FEBS Lett., 255, 405) succeeded in the purification of vitamin D
3
24-hydroxylase from rat kidney mitochondria extracted after administration of vitamin D. Subsequently Ohyama et al. (1991, FEBES Lett., 278, 195) isolated a cDNA by the screening of clones using an anti-vitamin D
3
24-hydroxylase antibody. It is known that this vitamin D
3
24-hydroxylase cDNA has a full length of 3.2 K bases and contains a reading frame (generally called open reading frame) of 1542 bp for the translation of 514 amino acids and produces a protein having a molecular weight of 59,000. It has been shown that as 35 amino acids are cleaved off from the N-terminus of the above protein, a mature protein consisting of 479 amino acid residues and having a molecular weight of about 55,000 is produced. Moreover, the finding that its 462nd amino acid cysteine is bound to the 5-position of heme indicates that this protein has characteristics similar to those of mitochondrial protein P-450, and an expression experiment in COS cells gave evidence of expression as a protein. Meanwhile, human vitamin D
3
24-hydroxylase cDNA has been isolated by Chen et al. (1993, ProNAS, USA, 90, 4543), the murine counterpart by Itoh et al. (1995, Biochemica et Biophysica Acta, 1264, 26), and guinea pig counterpart by Ohyama et al. (1996, Journal of Japan Society of Bone Metabolism, 14, 112). It has been shown that the amino acid sequences of the above rat, human, mouse and guinea pig versions of the enzyme have about 80 to 95% homology.
The functions of the enzyme in vivo has also been analyzed, and Shinki et al. (1992, J. Biol. Chem., 267, 13757) demonstrated by Northern analysis that vitamin D
3
24-hydroxylase is induced by 1&agr;,25-dihydroxyvitamin D
3
. It was also shown that the reaction of 1&agr;,25-dihydroxyvitamin D
3
is faster and higher in degree in the small intestine than in the kidneys. Furthermore, in the analysis of the relative reactivity of 25-hydroxyvitamin D
3
and 1&agr;,25-dihydroxyvitamin D
3
, the difference in Km value suggested that 1&agr;,25-dihydroxyvitamin D
3
is higher in the substrate specificity of vitamin D
3
24-hydroxylase. Bechen, M. J. et al. (1996, Biochemistry, 35, 8465) investigated the metabolism of 25-hydroxyvitamin D
3
experimentally in
Spodoptera frugiperda
(Sf21) cells and suggested that the enzyme has 23-/24-bicatalytic activity. Furthermore, vitamin D
3
25-hydroxylase has been isolated from rat liver mitochondria by Masumoto et al. (1988, J. Biol. Chem., 263, 14256) and some light has been cast on the pertinent gene function as well. However, neither isolation and purification nor cloning of vitamin D
3
1&agr;-hydroxylase gene has been successful to this day.
Expression of vitamin D
3
24-hydroxylase gene is regulated by the coupling of the heterodimer consisting of the vitamin D
3
receptor (generally abbreviated as VDR)-1&agr;,25-dihydroxyvitamin D
3
complex and the retinoid X receptor (generally
Isaka Masami
Kasuga Hisao
Matsuoka Kunio
Chao Mark
Clark Deborah J. R.
Nikodem David
Ramesh Elaine M.
Takeda Chemical Industries, Inc.
LandOfFree
25-hydroxyvitamin D3 24-hydroylase transgenic rats does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with 25-hydroxyvitamin D3 24-hydroylase transgenic rats, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and 25-hydroxyvitamin D3 24-hydroylase transgenic rats will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2577123