Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
2000-02-16
2002-04-02
Nguyen, Dave T. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S018000, C800S025000, C800S021000, C435S014000, C435S325000, C435S455000, C435S463000
Reexamination Certificate
active
06365796
ABSTRACT:
BACKGROUND OF THE INVENTION
Uncoupling protein 2 (UCP2) (Fleury, C., et al.,
Nat. Genet,
15:269 (1997); Gimeno, R. E., et al.,
Diabetes,
46:900 (1997)) and uncoupling protein 3 (UCP3) (Boss, O., et al.,
FEBS Lett.,
408:39 (1997); Vidal-Puig, A., et al.,
Biochem. Biophys. Res. Commun.,
235:79 (1997); Gong, D. W., et al.,
J. Biol. Chem.
272:24129 (1997)) are recently discovered members of the mitochondrial inner membrane carrier family with high homology to UCP1 (Nicholls, D. G., et al.,
Physiol. Rev.,
64:1 (1984); Klingenberg, M., and Huang, S. G.,
Biochim. Biophys. Acta.,
1415:271 (1999)) and expression patterns which are consistent with the hypothesis that they play a role in the regulation of cellular processes in which ATP plays a regulatory function. Consistent with this theory, studies in which UCP2 and UCP3 have been overexpressed in yeast (Rial, E., et al.,
EMBO J,
18:5827 (1999); Hinz, W., et al.,
FEBS Lett,
448:57 (1999); C. Y Zhang, et al.,
FEBS Lett,
449:129 (1999)) or reconstituted into proteoliposomes (Jaburek, M., et al.,
J Biol. Chem.,
274:26003 (1999)) indicate a proton leak (and as a consequence modulator of ATP) role for these new UCPs. UCP3 is expressed primarily in skeletal muscle where it likely plays a role in regulated thermogenesis. In contrast, UCP2 has a nearly ubiquitous expression pattern, but at varying levels in a number of tissues and cell types including tissues involved in glucose homeostasis (pancreatic islets, white fat, brown fat, heart, skeletal muscle). For example, UCP2 mRNA (Zhou, Y. T., et al.,
Proc. Natl. Acad. Sci. U.S.A.,
94:6386 (1997); Chan, C. B., et al.,
Diabetes
48:1482 (1999)) and protein are highly expressed in pancreatic &bgr;-cells.
&bgr;-cell function deteriorates in many individuals with obesity and insulin resistance, culminating in the development of type II diabetes mellitus. UCP2 mRNA expression is increased in adipose tissue of ob/ob obese mice, raising the possibility that it may also be increased in &bgr;-cells as well. If true, obesity-induced UCP2 expression in &bgr;-cells could contribute to &bgr;-cell dysfunction, promoting the development of diabetes. Consistent with this theory, it has been reported that UCP2 lies within a major quantitative trait loci (QTL) (murine chromosome 7; rat chromosome 1 and human chromosome 11) controlling diet-induced hyperinsulinemia in C57B1/6 mice (Fleury, C., et al.,
Nat. Genet,
15:269 (1997); Seldin, M. F., et al.,
J. Clin. Invest.,
94:269 (1994)); glucose intolerance and adiposity in the GK (Goto-Kakizaki) model of type 2 diabetes the rat (Gauguier, D., et al.,
Nat. genet.,
12:38 (1996); Galli, J., et al.,
Nat. genet.,
12:31 (1996); Kaisaki, P. J., et al.,
Mamm. genome,
9:910 (1998)), and human insulin-dependent diabetes locus-4 (Fleury, C., et al.,
Nat. Genet,
15(3):269-272 (1997)). Unfortunately, little is known about regulation of &bgr;-cell UCP2 gene expression during the pathogenesis of NIDDM. Similarly, little is known about regulators of UCP2 activity, which could also be altered, contributing to &bgr;-cell dysfunction.
Thus, additional studies, particularly in vivo studies, are needed to elucidate the biochemical physiological functions of UCP2 and to identify molecular targets, regulators and therapeutic strategies for the treatment or prevention of diseases or conditions associated with UCP2-regulated cellular processes.
SUMMARY OF THE INVENTION
The present invention relates to a transgenic non-human mammal (e.g., mouse) which lacks a functional UCP2 gene (also referred to herein as transgenic non-human UCP2 knockout mammal or a UCP2 knockout mammal). The transgenic non-human mammal of the present invention can have at least one non-functional allele for the UCP2 gene. In one embodiment, the transgenic non-human mammal is characterized by a disruption of the UCP2 gene which is either a homozygous disruption or a heterozygous disruption. In a particular embodiment, the genome of the UCP2 knockout mammal comprises a disruption of a segment between introns 2 and 7 of the UCP2 gene. In another embodiment, the genome of the UCP2 knockout mammal comprises an insertion of an exogenous nucleic acid sequence into an exon of the UCP2 gene.
As a result of the disruption of the UCP2 gene, the transgenic knockout mammal of the present invention manifests a particular phenotype. In one embodiment, the UCP2 knockout mammal has altered insulin/glucose homeostasis. In a particular embodiment, the transgenic non-human mammal is characterized by increased glucose-stimulated insulin secretion.
The invention further provides a method of producing a transgenic non-human mammal which lacks a functional UCP2 gene. In this method, a targeting vector is introduced into an embryonic stem cell to produce a transgenic stem cell in which the UCP2 gene is disrupted. A transgenic embryonic stem cell which includes a disrupted UCP2 gene due to the integration of the targeting vector into its genome is then selected. The selected embryonic stem cell is introduced into a blastocyst, thereby forming a chimeric blastocyst; and the chimeric blastocyst is introduced into the uterus of a pseudopregnant mammal wherein the pseudopregnant mammal gives birth to a transgenic non-human mammal which lacks a functional UCP2 gene due to heterozygous disruption of the UCP2 gene. The method can further comprise breeding the transgenic non-human mammal which lacks a functional UCP2 gene due to a heterozygous disruption with a second mammal of the same species to generate F1 progeny having a heterozygous disruption of the UCP2 gene, thereby expanding the population of mammals having a heterozygous disruption of the UCP2 gene. The F1 progeny are then crossbred to produce a transgenic non-human mammal which lacks a functional UCP2 gene due to a homozygous disruption of the UCP2 gene.
The present invention also relates to constructs or vectors (e.g., UCP2 targeting construct) designed to disrupt the function of a wild type mammalian UCP2 gene. In one embodiment, the invention provides a construct which comprises about 5.5 kb of a UCP2 sequence which is 5′ of an expression cassette. In a particular embodiment, the construct comprises 8.7 kb of genomic UCP2 sequence wherein 5.5 kb of the 8.7 kb genomic sequence is 5′ of an expression cassette and 3.2 kb of the 8.7 kb genomic sequence is 3′ of the expression cassette. More specifically, the invention provides a UCP2 gene replacement vector in which the genomic nucleotide sequence of the UCP2 gene between introns 2 and 7 is removed and/or replaced with a PGK-Neo-Poly(A) expression cassette.
The present invention also provides cells, cell lines, mammalian tissues, cellular extracts, organelles (e.g., mitochondria) and organs which lack a functional UCP2 gene. In one embodiment, the cells are pancreatic beta cells.
The cells, cell lines, mammalian tissues, cellular extracts, organelles (e.g., mitochondria) and organs of the instant invention can be used in a method for determining whether an agent inhibits UCP2. For example, pancreatic tissue, islets or cells isolated from wild-type mouse and a UCP2 knockout mouse can be used in combination to identify an agent which inhibits UCP2-mediated negative regulation of &bgr;-cell secretion of insulin. In one embodiment, a suitable in vitro screening method comprises combining cells which comprise a wild type UCP2 gene (wild type cells), an amount of glucose sufficient to stimulate insulin production and the agent; and combining cells which lack a functional UCP2 gene (UCP2 knockout cells), an amount of glucose sufficient to stimulate insulin production of the cells and the agent. The cells are maintained under conditions appropriate to stimulate insulin production. The amount of insulin produced by the wild type cells is compared to the amount of insulin produced by the UCP2 knockout cells, wherein if the amount of insulin produced by the wild type cells is increased compared to the amount of insulin produced by the knockout cells, then the agent inhibits UCP2.
The in vitro scre
Chan Catherine B.
Lowell Bradford B.
Wheeler Michael B.
Zhang Chen-Yu
Beth Israel Deaconess Medical Center
Hamilton Brook Smith & Reynolds P.C.
Nguyen Dave T.
Nguyen Quang
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