Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-08-04
2002-06-11
Mertz, Prema (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S324000, C435S069500, C435S071100, C435S071200, C435S320100, C435S471000, C435S325000, C435S252300, C435S254110
Reexamination Certificate
active
06403782
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to regulation of the immune system.
BACKGROUND OF THE INVENTION
The chemokines are a family of 8-12 kD proteins that regulate leukocyte trafficking by binding to specific seven transmembrane spanning G-protein-linked receptors. They can be divided into three families depending upon the sequence of conserved cysteine residues and this structural distinction corresponds to specific biologic properties in that the C-X-C, C-C, and C families are mainly chemoattractive for neutrophils, monocytes, and lymphocytes, respectively. Additionally, each chemokine family maps to a different chromosomal locus.
Eosinophils are circulating leukocytes that survive for several weeks. They dwell predominantly in tissues where they mediate pro-inflammatory and cytotoxic damage in selected diseases (e.g. asthma, parasitic infections, and malignancy). Given their presumed role in the pathogenesis of inflammatory states, the regulation of tissue recruitment of eosinophils is of interest and various chemoattractants have been found to be active on eosinophils, including leukotriene B4, platelet activating factor (PAF), and several chemokines (Resnick, et al. (1993) Amer. J. Resp. Cell. Mol. Biol. 8, 349-355). Chemokines active on eosinophils include certain C-C chemokines: monocyte chemoattractive protein (MCP)-2 and 3, RANTES, and macrophage inflammatory protein (MIP)-1a (Rot, et al. (1992) J. Exp. Med 176, 1489-1495; Alam, R. et al. (1993) J. Immun. 150, 3442-3448; Dahinden, et al. (1994) J. Exp. Med. 179, 751-756; Weber, et al. (1995) J. Immun. 154, 4166-4172). A C-X-C chemokine, interleukin-8 (IL-8), is also chemoattractive for cytokine-primed eosinophils (Warringa et al. (1993) J. All. Clin. Immun. 91, 1198-1205). Notwithstanding their activity, none of these chemoattractive molecules are eosinophil specific and their relative importance in selected diseases and in experimental animal models of allergy remains unclear.
SUMMARY OF THE INVENTION
In general, the invention features substantially pure nucleic acid (for example, genomic DNA, cDNA, or synthetic DNA, or mRNA) encoding an eotaxin polypeptide as defined below. In related aspects, the invention also features a vector, a cell (e.g., a bacterial, yeast, nematode, or mammalian cell), a transgenic animal which includes such a substantially pure DNA encoding an eotaxin polypeptide of the invention or a knockout mutation in the eotaxin gene, and methods for modulating eosinophil chemotaxis.
In preferred embodiments, the eotaxin gene is the human eotaxin gene provided in
FIG. 12
, the murine eotaxin gene provided in
FIG. 3A
or the guinea pig eotaxin gene provided in FIG.
7
. In various preferred embodiments, the cell is a transformed animal cell such as a human cell or a rodent cell.
In related aspects, the invention features a transgenic animal containing a transgene which encodes an eotaxin polypeptide and use of the eotaxin nucleotide sequence to engineer a transgenic animal having a knockout mutation in the eotaxin gene. The invention also features a cell that expresses the eotaxin gene. Preferably, the cell is an animal cell which is an epithelial or endothelial cell.
In a second aspect, the invention features a substantially pure DNA which includes a promoter capable of expressing the eotaxin gene in a cell. In preferred embodiments, the promoter is the promoter native to an eotaxin gene. Additionally, transcriptional and translational regulatory regions are preferably native to an eotaxin gene. A constitutive promotor or an inducible promotor are also included in the invention.
In other aspects, the invention features a substantially pure oligonucleotide including one or a combination of the sequences shown in
FIGS. 3A
(SEQ ID NO:5), 7(SEQ ID NO:15),
12
A (SEQ ID NO:17),
12
B (SEQ ID NO:19), and
12
C (SEQ ID NO:21).
In a another aspect, the invention features a method of isolating an eotaxin gene or fragment thereof from a cell, involving: (a) providing a sample of cellular DNA; (b) providing a pair of oligonucleotides having sequence homology to a conserved region of an eotaxin gene (for example, oligonucleotides which include fragments of the sequences shown in
FIGS. 3A
(SEQ ID NO:5), 7 (SEQ ID NO:15),
12
A (SEQ ID NO:17),
12
B (SEQ ID NO:19), and
12
C (SEQ ID NO:21) which are conserved as evidenced by the homologies shown in
FIG. 3B
(SEQ ID NOS.7-14(c) combining the pair of oligonucleotides with the cellular DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and (d) isolating the amplified eotaxin gene or fragment thereof. Where a fragment is obtained by PCR, standard library screening techniques may be used to obtain the complete coding sequence.
In preferred embodiments, amplification is carried out using a reverse-transcription polymerase chain reaction, for example, the RACE method.
In another aspect, the invention features a method of identifying a eotaxin gene in a cell, involving: (a) providing a preparation of cellular DNA (for example, from the human genome); (b) providing a detectably-labelled DNA sequence (for example, prepared by the methods of the invention) having homology to a conserved region of an eotaxin gene; (c) contacting the preparation of cellular DNA with the detectably-labelled DNA sequence under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (d) identifying an eotaxin gene by its association with the detectable label.
In another aspect, the invention features a method of isolating an eotaxin gene from a recombinant DNA library, involving: (a) providing a recombinant DNA library; (b) contacting the recombinant DNA library with a detectably-labelled gene fragment produced according to the PCR method of the invention under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (c) isolating an eotaxin gene by its association with the detectable label.
In another aspect, the invention features a method of isolating an eotaxin gene from a recombinant DNA library, involving: (a) providing a recombinant DNA library; (b) contacting the recombinant DNA library with a detectably-labelled Eotaxin oligonucleotide of the invention under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (c) isolating an eotaxin gene by its association with the detectable label.
In another aspect, the invention features an eotaxin gene isolated according to the method involving: (a) providing a sample of cellular DNA; (b) providing a pair of oligonucleotides having sequence homology to a conserved region of an eotaxin gene; (c) combining the pair of oligonucleotides with the cellular DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and (d) isolating the amplified eotaxin gene or fragment thereof.
In another aspect, the invention features an eotaxin gene isolated according to the method involving: (a) providing a preparation of cellular DNA; (b) providing a detectably-labelled DNA sequence having homology to a conserved region of an eotaxin gene; (c) contacting the preparation of DNA with the detectably-labelled DNA sequence under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (d) identifying an eotaxin gene by its association with the detectable label.
In another aspect, the invention features an eotaxin gene isolated according to the method involving: (a) providing a recombinant DNA library; (b) contacting the recombinant DNA library with a detectably-labelled eotaxin gene fragment produced according to the method of the invention under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (c) isolating an eotaxin gene by its association with the detectable label.
In another aspect, the invention features a method of identifying an eotaxin gene involving: (a) providing a mammalian cell sample; (b) introducing by transformation (e.g. viral, chemical, or mec
Garcia Eduardo
Leder Philip
Luster Andrew D.
Rothenberg Marc
Bieker-Brady, Ph.D. Kristina
Clark & Elbing LLP
Mertz Prema
President and Fellows of Harvard College
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