Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-02-10
2001-07-03
Clark, Deborah J. R. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S021000, C435S320100
Reexamination Certificate
active
06255556
ABSTRACT:
BACKGROUND OF THE INVENTION
Obesity is a significant risk factor for many serious illnesses, such as heart disease, arthritis, and diabetes. To better understand the mechanisms that lead to obesity and to develop strategies to control certain types of obesity, a better knowledge of the molecular events that regulate fat cell differentiation and fat storage is needed at the organismal level. Transgenic animals with altered fat metabolism provide a unique opportunity to probe the relationships between gene expression and fat metabolism.
SUMMARY OF THE INVENTION
The invention is based on a new transgenic animal that contains a CCAAT/enhancer-binding protein (C/EBP) &agr; gene in which the open reading frame encoding the C/EBP&agr; polypeptide has been replaced with a C/EBP&bgr; open reading frame. This animal exhibits decreased accumulation of fat in white adipose tissue (WAT). Thus, the transgenic animals of the invention offers a unique animal model in which the genetic and biochemical mechanisms responsible for decreased WAT mass can be deciphered.
Accordingly, the invention features a transgenic animal (e.g., a non-human mammal, rodent, rat, mouse, rabbit, pig, cow, chicken, or fish) whose genomic DNA includes a gene having a C/EBP&agr; promoter operably linked to a DNA sequence encoding a C/EBP&bgr; polypeptide. By “operably linked” is meant that a nucleotide sequence (e.g., an open reading frame) is linked to a regulatory sequence (e.g., a promoter) in a manner which allows for expression of the nucleotide sequence in vitro or in vivo. The transgenic animal exhibits reduced accumulation of (e.g., a statistically significant reduction in) fat in its WAT, as compared to a reference animal (e.g., the wild-type animal from which the transgenic animal was generated) whose genomic DNA does not contain the gene.
A “C/EBP&agr;” promoter refers to a promoter that directs transcription of an RNA in a temporal and cell-type specific manner at least substantially similar, if not identical, to the expression of a wild-type C/EBP&agr; gene. Thus, a “C/EBP&agr;” promoter includes wild-type C/EBP&agr; promoters as well as such promoters containing insertions, deletions, or substitutions that do not affect the temporal and tissue-specific expression of the C/EBP&agr; promoter. Similarly, a “C/EBP&bgr; polypeptide” is a polypeptide that has at least one biochemical or cellular activity associated with a wild-type C/EBP&bgr; polypeptide. Thus, a C/EBP&bgr; polypeptide includes wild-type C/EBP&bgr; polypeptides as well as such polypeptides containing amino acid insertions, deletions, or substitutions that do not affect the biochemical or cellular activity of the C/EBP&bgr; polypeptide.
The genomic DNA of the transgenic animal can also contain multiple copies (i.e., two or more copies) of the gene, so long as the transgenic animal exhibits reduced accumulation of fat in its white adipose tissue. In addition, when the gene is made by replacing a portion of a wild-type endogenous C/EBP&agr; gene with a C/EBP&bgr; open reading frame, the transgenic animal can be homozygous or heterozygous for the mutated C/EBP&agr; gene.
The transgenic animal of the invention can also include one or more of the following phenotypes: the size of adipocytes in the WAT of the transgenic animal is smaller than the size of adipocytes in the WAT of the reference animal; the serum glucose and triacylglyceride levels in the transgenic animal are substantially the same (i.e., with no statistically significant difference) as those in the reference animal; the transgenic animal consumes more food than the reference animal, provided that the amount of food available is not limiting and the same type of food is presented to the transgenic and reference animals; reduced accumulation of fat is observed when the transgenic and reference animals are fed a diet that is at least about 30% by weight fat; the expression level of adipsin in the white adipose tissue of the transgenic animal is reduced as compared to the expression level of adipsin in the white adipose tissue of the reference animal; the expression level of leptin in the white adipose tissue of the transgenic animal is reduced as compared to the expression level of leptin in the white adipose tissue of the reference animal; the total mass of brown adipose tissue in the transgenic animal is greater than the total mass of brown adipose tissue in the reference animal; and the mitochondrial content in the white adipose tissue of the transgenic animal is greater than the mitochondrial content in the white adipose tissue of the reference animal. The terms “smaller,” “reduced,” “more,” or “greater” can refer to differences that are statistically significant.
The invention also features a method of producing a transgenic animal of the invention by replacing at least a portion of a C/EBP&agr; gene in the genomic DNA of the transgenic animal with a DNA insert comprising a first sequence encoding a C/EBP&bgr; polypeptide and a second sequence encoding a selectable marker; and removing the second sequence from the genomic DNA, thereby producing the transgenic animal.
In addition, the invention includes a nucleic acid comprising a C/EBP&agr; promoter operably linked to a sequence encoding C/EBP&bgr; polypeptide, which is useful for producing a transgenic animal of the invention.
The transgenic animals of the invention can be used to elucidate the biochemical and genetic determinants that provide for a lean (low fat) body. For example, cDNA from white adipose tissue of the transgenic and reference animal can be interrogated with DNA chips to uncover any differences in expression profiles. Individual hits, or genes showing significant changes in expression, may be or encode for potential drugs or drug targets for decreasing body fat.
Other features or advantages of the present invention will be apparent from the following detailed description and also from the claims.
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Ebert, et al., 1988, Molecular Endocrinology, 2:277-83.*
Mullins, et al., 1996, 98(11):S37-S40.*
Freytag, 1994, Genes and Development, 8:1654-63.*
Cowherd et al., “Molecular regulation of adipocyte differentiation,” Cell & Developmental Biology, vol. 10, pp. 3-10, 1999.
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Spiegelman et al., “Adipogenesis and Obesity: Rounding Out the Big Picture,” Cell, vol. 87, pp. 277-389, 1996.
Wang et al., “Impaired Energy Homeostasis in C/EBP&agr; Knockout Mice,” Science, vol. 269, pp. 1108-1112, 1995.
Lee et al., “Disruption of the c/ebp&agr; Gene in Adult Mouse Liver,” Molecular and Cellular Biology, vol. 17, pp. 6014-6022, 1997.
Academia Sinica
Clark Deborah J. R.
Fish & Richardson P.C.
Sorbello Eleanor
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