WO1998042826A1 - Tissue specific promoters - Google Patents
Tissue specific promoters Download PDFInfo
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- WO1998042826A1 WO1998042826A1 PCT/US1998/005866 US9805866W WO9842826A1 WO 1998042826 A1 WO1998042826 A1 WO 1998042826A1 US 9805866 W US9805866 W US 9805866W WO 9842826 A1 WO9842826 A1 WO 9842826A1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
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- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A—HUMAN NECESSITIES
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- A01K2217/00—Genetically modified animals
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
Definitions
- the present invention pertains to the identification and characterization of a nucleic acid sequence of the human involucrin gene which targets expression of any desired nucleic acid sequence to specific tissues and specific cells.
- this invention relates to nucleic acid sequences which target expression of nucleic acid sequences to suprabasal cells in stratifying squamous epithelial tissue and to uroepithelial cells.
- this invention pertains to transgenic animals which exhibit cancer of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung, as well as epidermal and ectocervical hyperplasias.
- this invention pertains to methods of screening for therapeutics for epithelial neoplasia.
- epithelial cells are the single most common cause of morbidity and mortality of humans. Foremost among these diseases is cancer.
- Other diseases which are epithelial in origin include, for example, blistering disease (e.g., epidermolytic hyperkeratosis, and Dowling-Meara disease) proliferative disease (e.g., psoriasis, epidermal lysis, and Bulosa simplex) and Ichthyosis disease (e.g., Ichthyosis bullosa Simens, and recessive X-linked ichthyosis).
- blistering disease e.g., epidermolytic hyperkeratosis, and Dowling-Meara disease
- proliferative disease e.g., psoriasis, epidermal lysis, and Bulosa simplex
- Ichthyosis disease e.g., Ichthyosis bullosa Simens, and recessive X-
- epithelium as the lining of tissue surfaces in the body places it at a particularly high risk for repeated damage from a variety of agents in the environment.
- epithelial cancers e.g., cancer of the lung, breast, colon, liver, cervix, etc.
- carcinogens such as cigarette smoke, hydrocarbons in grilled foods, toxic molds, and infection with genital DNA tumor viruses.
- candidate therapeutics directed at the treatment of epithelial disease has traditionally focused on animal models in which the animal is repeatedly exposed to one or a combination of chemicals. For example, models for cancer development and treatment rely on administration of carcinogenic and co-carcinogenic compounds.
- transgenic animal models which develop cancer and in which selected genes are expressed in epithelial cells in general [e.g., U.S. Patent No. 5,550,316; Griep et al. (1994) Proc. Soc. Exp. Biol. Med. 206:24-34; Kondoh et al. (1995) Intervirology 38:181-186; Yang et al. (1995) Am J. Pathol. 147:68-78; Greenhalgh et al. (1994) Cell Growth Differ. 5:667-675; Tinsley et al. (1992) J. Gen. Virol. 73:1251-1260] have been described.
- HPV human papillomavirus
- transgenic animals which develop epithelial cell disease in general, and neoplastic and/or preneoplastic lesions in particular, there is no transgenic model for some epithelial diseases (e.g., blistering disease, proliferative disease, and Ichthyosis disease) or for certain cancers (e.g., colon cancer, anal cancer, etc.).
- epithelial diseases e.g., blistering disease, proliferative disease, and Ichthyosis disease
- cancers e.g., colon cancer, anal cancer, etc.
- a single transgenic animal model is of limited use in comprehensive screening of therapeutics. This is because a compound which is not therapeutic in a transgenic animal that has a particular genetic alteration, may nevertheless be therapeutic in a transgenic animal which develops the same disease as a result of a different genetic alteration.
- the present invention provides methods for selective expression of a nucleic acid sequence of interest in epithelial cells of a non-human transgenic animal, and in particular to suprabasal epithelial cells.
- This invention further relates to methods for producing a non- human transgenic animal wherein a nucleotide sequence of interest is selectively expressed in epithelial cells of the non-human animal, and more particularly in suprabasal epithelial cells.
- the present invention also relates to the use of the transgenic animals for screening anti- neoplastic compounds. Further provided by this invention are oligonucleotide sequences which selectively target expression of a nucleotide sequence of interest to epithelial cells, and in particular to suprabasal epithelial cells.
- the present invention provides a purified oligonucleotide comprising at least a portion of the nucleotide sequence of SEQ ID NO:l. While it is not intended that the present invention be limited to a particular type of activity of the portion of the nucleotide sequence of SEQ ID NO:l, in one embodiment, the portion of oligonucleotide is characterized by having promoter activity. Furthermore, while it is not contemplated that the invention be limited to a particular portion of SEQ ID NO:l, in a preferred embodiment, the portion of SEQ ID NO:l comprises the nucleotide sequence from -1953 to -1 of SEQ ID NO:l or variants or homologs thereof.
- the portion of SEQ ID NO:l comprises the nucleotide sequence from -1333 to -1 of SEQ ID NO:l or variants or homologs thereof. In yet another embodiment, the portion of SEQ ID NO:l comprises the nucleotide sequence from -986 to -1 of SEQ ID NO:l or variants or homologs thereof.
- the portion of the nucleotide sequence is operably linked to a nucleic acid sequence of interest.
- the invention is contemplated not to be limited to the type or nature of the nucleic acid sequence which is operably linked to the nucleotide sequence of the invention.
- the promoter activity of the portion of the nucleotide sequence of SEQ ID NO:l is tissue specific. While not intending to limit the invention to a particular type of tissue, in one embodiment, the tissue is selected from the group consisting of uroepithelial tissue and stratified squamous epithelial tissue. In a preferred embodiment, it is contemplated that the stratified squamous epithelial tissue is in an organ selected from the group consisting of epidermis and cervix. In yet a more preferred embodiment, the stratified squamous epithelial tissue specific promoter activity is cell type specific. In a further preferred embodiment, it is contemplated that the cell in the stratified squamous epithelial tissue is suprabasal.
- the present invention also provides a recombinant expression vector comprising at least a portion of the nucleotide sequence of SEQ ID NO:l. While it is not intended that the present invention be limited to the type of activity of the portion of the nucleotide sequence of SEQ ID NO:l, in one embodiment, the portion of the oligonucleotide is characterized by having promoter activity. Furthermore, while it is not contemplated that the invention be limited to a particular portion of SEQ ID NO:l, in a preferred embodiment, the portion of SEQ ID NO:l comprises the nucleotide sequence from -1953 to -1 of SEQ ID NO:l or variants or homologs thereof.
- the portion of SEQ ID NO:l comprises the nucleotide sequence from -1333 to -1 of SEQ ID NO:l or variants or homologs thereof. In yet another embodiment, the portion of SEQ ID NO:l comprises the nucleotide sequence from -986 to -1 of SEQ ID NO:l or variants or homologs thereof.
- the portion of the nucleotide sequence is operably linked to a nucleic acid sequence of interest. It is not intended that the invention be limited to the type or nature of the nucleic acid sequence which is operably linked to the nucleotide sequence of the invention.
- a host cell comprising a recombinant expression vector wherein the recombinant expression vector comprises at least a portion of the nucleotide sequence of SEQ ID NO:l. While it is not intended that the host cell be limited to a particular cell type, in a preferred embodiment, the host cell is a fertilized egg cell. In an alternative preferred embodiment, the host cell is in a blastomere. In a further preferred embodiment, the host cell is in an eight-cell embryo. In yet another preferred embodiment, the host cell is in a midgestation embryo. In yet a further preferred embodiment, the host cell is an embryonic stem cell.
- the present invention further provides a transgenic non-human animal capable of tissue specific expression of a nucleic acid sequence of interest, wherein the transgenic non- human animal comprises an oligonucleotide comprising at least a portion of the nucleotide sequence of SEQ ID NO:l operably linked to the nucleic acid sequence of interest.
- expression takes place in a tissue selected from the group consisting of stratified squamous epithelial tissue and uroepithelial tissue.
- the stratified squamous epithelial tissue is in an organ selected from the group consisting of epidermis and cervix.
- the nucleic acid sequence of interest is a coding sequence of an oncogene.
- the oncogene is a human papillomavirus 16 oncogene.
- the transgenic non-human animal is characterized by having cancer in a tissue selected from the group consisting of tracheal, esophageal, colon, epidermal, anal, rectal, lymph node, spleen, and lung tissue.
- the transgenic non-human animal is further characterized by having hyperplasia in a tissue selected from the group consisting of epidermal and cervical tissue.
- Also provided by the invention is a method for selective expression of a nucleic acid sequence of interest in epithelial cells of a non-human animal, comprising: a) providing: i) a transgene, wherein the transgene contains at least a portion of the nucleotide sequence of SEQ ID NO:l operably linked to the nucleic acid sequence of interest; ii) an embryonic cell of a non-human animal; and iii) a pseudopregnant non-human animal; b) introducing: i) the transgene into the embryonic cell to produce a transgenic embryonic cell; and ii) the transgenic embryonic cell into the pseudopregnant non-human animal under conditions such that the pseudopregnant non-human animal delivers progeny derived from the transgenic embryonic cell, wherein the nucleic acid sequence of interest is selectively expressed in the epithelial cells of the progeny.
- the method of the invention further comprises c) identifying at least one offspring of the progeny wherein the nucleic acid sequence of interest is selectively expressed in the epithelial cells of the offspring.
- the portion consists of the nucleotide sequence from -1333 to -7 of SEQ ID NO:l. In an alternative embodiment, the portion consists of the nucleotide sequence from -986 to -7 of SEQ ID NO:l.
- the present invention also provides a method for producing a non-human transgenic animal, comprising: a) providing: i) a transgene, wherein the transgene contains at least a portion of the nucleotide sequence of SEQ ID NO:l operably linked to one or more oncogenes; ii) an embryonic cell of a non-human animal; and iii) a pseudopregnant non- human animal; b) introducing: i) the transgene into the embryonic cell to produce a transgenic embryonic cell; and ii) the transgenic embryonic cell into the pseudopregnant non-human animal under conditions such that the pseudopregnant non-human animal delivers progeny derived from the transgenic embryonic cell; and c) identifying at least one offspring of the progeny, wherein the oncogne is selectively expressed in epithelial cells of the offspring.
- the epithelial cell is suprabasal. While not intending to limit the oncogene to a particular oncogene, in one embodiment, the oncogene consist of human papillomavirus 16 oncogne E6 nucleic acid sequence and oncogne E7 nucleic acid sequence.
- the non-human transgenic animal is further characterized by having cancer in one or more tissues selected from the group comprising trachea, esophagus, colon, epidermis, anus, rectum, lymph node, spleen and lung. In yet another preferred embodiment, the non- human transgenic animal is further characterized by having hyperplasia in one or more tissues comprising epidermis and cervix.
- the present invention further provides a method of screening anti-neoplastic compounds, comprising: a) providing: i) a transgenic non-human animal having cancer, wherein the transgenic non-human animal contains a DNA sequence comprising at least a portion of the nucleotide sequence of SEQ ID NO:l or variants or homologs of the nucleotide sequence; and ii) a compound suspected of having anti-neoplastic activity; b) administering the compound to the transgenic non-human animal to produce a treated transgenic non-human animal; and c) detecting anti-neoplastic activity in the treated transgenic non-human animal, thereby identifying the compound as anti-neoplastic.
- the cancer is colon cancer.
- the cancer is anal cancer.
- Figure 1 shows the nucleic acid sequence (SEQ ID NO:l) of the hINV sequence from position -2473 to -1.
- Figure 2 shows the nucleotide genomic sequence (SEQ ID NO:6) of the sense DNA strand of HPV 16.
- Figure 3 shows a diagrammatic representation of the generation of the pINV
- Figure 4 shows a Southern blot of tail DNA from HPV 16 E6/E7 transgenic mice.
- Figure 5 shows a map of the E16E, H6B, Ha5.5B, A4.3B, and K4B constructs.
- Figure 6 shows a Western blot of hINV protein expression in the epidermis of non- transgenic (NT) mice and mice transgenic for the E16E construct (E16E) and the H6B construct (H6B).
- nucleic acid sequence refers to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
- nucleotide sequence of interest refers to any nucleotide sequence, the manipulation of which may be deemed desirable for any reason, by one of ordinary skill in the art.
- nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and of regulatory genes (e.g., activator protein 1 (API), activator protein 2 (AP2), Spl, etc.).
- regulatory genes e.g., activator protein 1 (API), activator protein 2 (AP2), Spl, etc.
- nucleotide sequences include non-coding regulatory elements which do not encode an mRNA or protein product, such as for example, a promoter sequence, an enhancer sequence, etc.
- amino acid sequence and polypeptide sequence are used interchangeably herein to refer to a sequence of amino acids.
- a "variant" of a first nucleotide sequence is defined as a nucleotide sequence which differs from the first nucleotide sequence e.g., by having one or more deletions, insertions, or substitutions that may be detected using hybridization assays or using DNA sequencing. Included within this definition is the detection of alterations to the genomic sequence of the first nucleotide sequence.
- hybridization assays may be used to detect alterations in (1) the pattern of restriction enzyme fragments capable of hybridizing to a genomic sequence of the first nucleotide sequence (i.e., RFLP analysis), (2) the inability of a selected portion of the first nucleotide sequence to hybridize to a sample of genomic DNA which contains the first nucleotide sequence (e.g., using allele-specific oligonucleotide probes), (3) improper or unexpected hybridization, such as hybridization to a locus other than the normal chromosomal locus for the first nucleotide sequence (e.g., using fluorescent in situ hybridization (FISH) to metaphase chromosomes spreads, etc.)].
- FISH fluorescent in situ hybridization
- a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
- An “insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to, for example, the naturally occurring nucleotide or amino acid sequence.
- substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
- portion when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence.
- the fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.
- oligonucleotide sequence which is a "homolog" of a first nucleotide sequence is defined herein as an oligonucleotide sequence which exhibits greater than or equal to 50% identity to the first nucleotide sequence when sequences having a length of 25 bp or larger are compared.
- hINV upstream nucleic acid sequence and "hINV upstream nucleotide sequence” refer to at least a portion of the nucleotide sequence comprising the nucleotide sequence from -2473 to -1 of Figure 1, and to variants, and homologs thereof.
- recombinant DNA molecule refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
- recombinant protein or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule.
- vector and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
- expression vector or "expression cassette” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
- Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
- Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
- operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
- the terms also refer to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
- transfection refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene- mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.
- the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "5'-
- CAGT-3' is complementary to the sequence "5'-ACTG-3 ⁇ "
- Complementarity can be “partial” or “total.”
- Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules.
- Total or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.
- the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
- nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity).
- a nucleotide sequence which is partially complementary, i.e., “substantially homologous,” to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay
- a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency -ire such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
- the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding the probe will not hybridize to the second non-complementary target.
- Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 «H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X
- Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
- low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target ( DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
- conditions which promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
- substantially homologous refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
- the term “substantially homologous” refers to any probe which can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
- hybridization is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex.
- Hybridization and the strength of hybridization is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
- hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
- the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
- a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support [e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
- a solid support e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
- T m is used in reference to the "melting temperature.”
- the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
- stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. "Stringency” typically occurs in a range from about T m °C to about 20°C to 25°C below T m . As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under “stringent conditions” the nucleotide sequence between positions -2473 and -1 (SEQ ID NO:l) ( Figure 1) or portions thereof will hybridize to its exact complement and closely related sequences.
- the stringent conditions include the choice of fragments of SEQ ID NO:l to be used. Fragments of SEQ ID NO:l which contain unique sequences (i.e., regions which are either non-homologous to or which contain less than 50% homology or complementarity with SEQ ID NO:l) are preferentially employed. Conditions of "weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually low between such organisms.
- amplifiable nucleic acid is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid” will usually comprise "sample template.”
- heterologous nucleic acid sequence or “heterologous DNA” are used interchangeably to refer to a nucleotide sequence which is ligated to a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
- Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.
- sample template refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest.
- background template is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
- PCR polymerase chain reaction
- the length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter.
- the method is referred to as the “polymerase chain reaction” (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.”
- PCR it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32 P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
- any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
- the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
- RNA is reverse transcribed using a single primer (e.g., an oligo-dT primer) prior to PCR amplification of the desired segment of the transcribed DNA using two primers.
- a single primer e.g., an oligo-dT primer
- the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and of an inducing agent such as DNA polymerase and at a suitable temperature and pH).
- the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
- the primer is an oligodeoxyribonucleotide.
- the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
- the term "probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest.
- a probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labelled with any
- reporter molecule so that it is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
- enzyme e.g., ELISA, as well as enzyme-based histochemical assays
- fluorescent, radioactive, and luminescent systems It is not intended that the present invention be limited to any particular detection system or label.
- restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double- or single-stranded DNA at or near a specific nucleotide sequence.
- DNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring.
- an end of an oligonucleotide is referred to as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring.
- a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
- discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' direction along the DNA strand.
- the promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region.
- enhancer elements can exert their effect even when located 3' of the promoter element and the coding region.
- Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.
- the term "hINV upstream sequence” refers to a sequence which is located 5' of the human involucrin gene transcription start site, as exemplified by SEQ ID NO:l depicted in Figure 1.
- an oligonucleotide having a nucleotide sequence encoding a gene means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product.
- the coding region may be present in either a cDNA, genomic DNA or RNA form.
- the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
- Suitable control elements such as enhancers, promoters, splice junctions, polyadenylation signals, etc.
- the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
- Enhancer elements consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis, T. et al, (1987) Science 236:1237]. Enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses. The selection of a particular enhancer depends on what cell type is to be used to express the protein of interest. The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site [Sambrook, J.
- a commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.
- Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.
- the term "poly A site” or "poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded.
- the poly A signal utilized in an expression vector may be "heterologous” or "endogenous.”
- An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
- a heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene.
- the term "promoter,” “promoter element,” or “promoter sequence” as used herein, refers to a DNA sequence which when placed at the 5' end of (i.e., precedes) an oligonucleotide sequence is capable of controlling the transcription of the oligonucleotide sequence into mRNA.
- a promoter is typically located 5' (i.e., upstream) of an oligonucleotide sequence whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and for initiation of transcription.
- human involucrin promoter and "hINV promoter” refer to a promoter sequence derived from the human involucrin gene. hINV promoter sequences are exemplified by, but not limit to, SEQ ID NO:l of Figure 1, the nucleotide sequence from -1953 to -1, from -1333 to -1 and from -986 to -1 of SEQ ID NO:l.
- promoter activity when made in reference to a nucleic acid sequence refers to the ability of the nucleic acid sequence to initiate transcription of an oligonucleotide sequence into mRNA.
- tissue specific refers to a promoter that is capable of directing selective expression of an oligonucleotide sequence to a specific type of tissue in the relative absence of expression of the same oligonucleotide in a different type of tissue.
- a promoter sequence located between positions - 2473 to -7 ( Figure 1) of the human involucrin gene is capable of directing selective expression of human involucirn gene sequences in epidermal, cervical, esophageal, tracheal, anal/rectal and oral tissues, and not in heart and liver tissues.
- Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of an animal such that the reporter construct is integrated into every tissue of the resulting transgenic animal, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic animal.
- the detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected.
- cell type specific refers to a promoter which is capable of directing selective expression of an oligonucleotide sequence in a specific type of cell in the relative absence of expression of the same oligonucleotide sequence in a different type of cell within the same tissue.
- a promoter sequence disclosed herein located between positions -2473 to -7 ( Figure 1) of the human involucrin gene is capable of directing selective expression of human involucirn gene sequences in uroepithelia cells of the kidney.
- cuboidal cells in the same kidney tissue did not express a heterologous involucrin gene sequence.
- cell type specific when applied to a promoter also means a promoter capable of promoting selective expression of an oligonucleotide in a region within a single tissue.
- the promoter sequence located between positions -2473 and -7 ( Figure 1) of the human involucrin gene directs expression of a gene to the suprabasal region of ectocervical epithelium, and not to the basal region of the ectocervical epithelium.
- the -2473 to -7 involucrin promoter sequence ( Figure 1) directs expression of a gene to the suprabasal region of epidermal epithelium, and not to the basal region of the epidermal epithelium.
- Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining as described herein. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody which is specific for the polypeptide product encoded by the oligonucleotide sequence whose expression is controlled by the promoter. A labeled (e.g., peroxidase conjugated) secondary antibody which is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.
- a labeled (e.g., peroxidase conjugated) secondary antibody which is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.
- selective expression refers to a comparison of relative levels of expression in two or more regions of interest.
- selective expression when used in connection with tissues refers to a substantially greater level of expression of a gene of interest in a particular tissue, or to a substantially greater number of cells which express the gene within that tissue, as compared, respectively, to the level of expression of, and the number of cells expressing, the same gene in another tissue.
- Selective expression does not require, although it may include, expression of a gene of interest in a particular tissue and a total absence of expression of the same gene in another tissue.
- selective expression refers to a substantially greater level of expression of, or a substantially greater number of cells which express, a gene of interest in a particular cell type, when compared, respectively, to the expression levels of the gene and to the number of cells expressing the gene in another cell type.
- nucleotide sequences are ligated in tandem either in the absence of intervening sequences, or in the presence of intervening sequences which do not comprise one or more control elements.
- transfection or "transfected” refers to the introduction of foreign DNA into a cell.
- nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
- antisense is used in reference to RNA sequences which are complementary to a specific RNA sequence (e.g., mRNA).
- Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced into a cell, this transcribed strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated.
- the term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the "sense" strand.
- the designation (-) (i.e., "negative") is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.
- the term "Southern blot” refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used.
- the DNA may be cleaved with restriction enzymes prior to electrophoresis.
- the DNA may be partially depurinated and denatured prior to or during transfer to the solid support.
- Southern blots are a standard tool of molecular biologists [J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58].
- Northern blot refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used.
- Northern blots are a standard tool of molecular biologists [J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52].
- reverse Northern blot refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane.
- the immobilized DNA is then probed with a labeled oligo-ribonucleotide probe or RNA probe to detect DNA species complementary to the ribo probe used.
- isolated when used in relation to a nucleic acid, as in "an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source.
- Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature.
- non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature.
- a given DNA sequence e.g., a gene
- RNA sequences such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins.
- isolated nucleic acid encoding a polypeptide of interest includes, by way of example, such nucleic acid in cells ordinarily expressing the polypeptide of interest where the nucleic acid is in a chromosomal or extrachromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
- the isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form.
- the oligonucleotide When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).
- the term "purified” or "to purify” refers to the removal of undesired components from a sample.
- the polypeptides are purified by the removal of host cell proteins thereby increasing the percent of recombinant polypeptides in the sample.
- substantially purified refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, more preferably 90% free, and most preferably 100% free from other components with which they are naturally associated.
- An "isolated polynucleotide” is therefore a substantially purified polynucleotide.
- coding region when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule.
- the coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG” which encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).
- hINV coding region refers to the sequence of exon 1 , intron 1, and exon 2 of the human involucrin gene, which is located in a EcoRI -restricted Charon 4A ⁇ I-3 [Eckert and Green (1986) Cell 46:583-589]).
- structural gene or "structural nucleotide sequence” refers to a DNA sequence coding for RNA or a protein.
- a “regulatory gene” or “regulatory sequence” is a structural gene which encodes products (e.g., transcription factors) which control the expression of other genes .
- regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
- a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
- Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
- the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene.
- a “gene” may also include non-translated sequences located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the length of the full-length mRNA.
- the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
- the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.
- the term “gene” encompasses both cDNA and genomic forms of a gene.
- a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
- Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
- mRNA messenger RNA
- genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript).
- the 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene.
- the 3' flanking region may contain sequences which direct the termination of transcription, post-transcriptional cleavage and polyadenylation.
- oncogene refers to a gene which is capable of transforming a normal cell to a cancer cell.
- An oncogene may be a viral oncogene or a cellular oncogene.
- a "viral oncogene” may be an early gene of a DNA virus (e.g., polyomavirus, papillomavirus, T-cell leukemia virus), or a cellular proto-oncogene incorporated into the genome of a transducing retroviruses such that the cellular proto-oncogene (e.g., c-src) is activated into an oncogene (e.g., v-src).
- a "cellular oncogene” is a mutated cellular gene formed in situ in the chromosome of a cell rather than introduced into the cell by a DNA virus or a transducing virus.
- cancer cell refers to a cell undergoing early, intermediate or advanced stages of multi-step neoplastic progression as previously described [H.C. Pitot (1978) in “Fundamentals of Oncology,” marcel Dekker (Ed.), New York pp 15-28]. The features of early, intermediate and advanced stages of neoplastic progression have been described using microscopy. Cancer cells at each of the three stages of neoplastic progression generally have abnormal karyotypes, including translocations, inversion, deletions, isochromosomes, monosomies, and extra chromosomes.
- a cell in the early stages of malignant progression is referred to as "hyperplastic cell” and is characterized by dividing without control and/or at a greater rate than a normal cell of the same cell type in the same tissue. Proliferation may be slow or rapid but continues unabated.
- a cell in the intermediate stages of neoplastic progression is referred to as a "dysplastic cell.”
- a dysplastic cell resembles an immature epithelial cell, is generally spatially disorganized within the tissue and loses its specialized structures and functions. During the intermediate stages of neoplastic progression, an increasing percentage of the epithelium becomes composed of dysplastic cells. "Hyperplastic” and “dysplastic” cells are referred to as "pre-neoplastic” cells.
- Neoplastic cells are typically invasive i.e., they either invade adjacent tissues, or are shed from the primary site and circulate through the blood and lymph to other locations in the body where they initiate secondary cancers.
- cancer or “neoplasia” refers to a plurality of cancer cells.
- epithelial cell refers to a cuboidal-shaped, nucleated cell which generally located on the surface of a tissue.
- a layer of epithelial cells generally functions to provide a protective lining and/or surface that may also be involved in transport processes.
- An epithelial cell is readily distinguished from a non-epithelial cell (e.g., muscle cell, nerve cell, etc.) using histological methods well known in the art.
- non-stratifying cell refers to an epithelial cell in a non-stratifying epithelial tissue.
- a “non-stratifying epithelial tissue” refers to a tissue which contains only a single layer of epithelial cells.
- Non-stratifying epithelial tissue is exemplified by, but is not limited to, epithelia lining the oviduct, gall bladder, kidney ducts, blood vessels, salivary gland ducts, pancreatic ducts, urinary tract lumen, etc.
- Non-stratifying epithelial tissue, stratifying epithelial tissue, and stratified squamous epithelial tissue tissue are readily distinguished one from the other by histological methods well known in the art, e.g., where tissue sections are stained with hematoxylin & eosin, or another stain.
- stratifying cell refers to an epithelial cell in a stratifying epithelial tissue.
- stratifying epithelial tissue refers to a tissue containing two or more layers of epithelial cells wherein the epithelial cells undergo morphological and functional changes.
- a "stratified squamous epithelial tissue” contains a basal layer of epithelial cells, a supra basal layer of epithelial cells and a surface layer of epithelial cells.
- the basal layer is proximal to the organ lined by the stratified squamous epithelial tissue, the surface layer is distal to the lined organ, whereas the suprabasal layer is located between the basal layer and the surface layers.
- stratified squamous epithelial tissue includes, but is not restricted to, ectocervix, vagina, epidermis, etc.
- squamous cell refers to an epithelial cell in a stratified squamous epithelial tissue.
- a squamous cell may be a basal cell, a suprabasal cell, or a surface cell.
- basal cell refers, respectively, to a squamous epithelial cell which is located in the basal layer, suprabasal layer, and surface layer of a stratified squamous epithelial tissue.
- a basal cell, suprabasal cell and surface cell are readily distinguishable on the basis of their morphology as determined, for example, by histochemical staining methods known in the art [e.g., Wheater et al., (1987) in "Functional Histology,” 2nd Edition, Churchill/Livingstone (Eds.) New York, 303, pp 65-70].
- Basal epithelial cells are generally cuboidal, suprabasal cells are generally less cuboidal and more flattened than adjacent basal cells, while surface cells are more flattened than both basal cells and suprabasal cells of the same stratified squamous epithelial tissue.
- Uroepithelial cell and “transitional epithelial cell” refer to an epithelial cell in the uroepithelial tissue.
- uroepithelial tissue refers to epithelial tissue located at the renal pelvic area where the ureter meets with the kidney.
- Uroepithlial cells are unique to the urine conducting passage of the urinary system and are characterized by having a thickened plasma membrane.
- non-human animal refers to any animal which is not a human and includes vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
- Preferred non-human animals are selected from the order Rodentia.
- the term "order Rodentia” refers to rodents i.e., placental mammals (class Euthria) which include the family Muridae (e.g., rats and mice), most preferably mice.
- transgenic animal refers to an animal that includes a transgene which is inserted into an embryonal cell and which becomes integrated into the genome either of somatic and/or germ line cells of the animal which develops from that embryonal cell, or of an offspring of such an animal.
- a "transgene” means a DNA sequence which is partly or entirely heterologous (i.e., not present in nature) to the animal in which it is found, or which is homologous to an endogenous sequence (i.e., a sequence that is found in the animal in nature) and is inserted into the animal's genome at a location which differs from that of the naturally occurring sequence.
- Transgenic animals which include one or more transgenes are within the scope of this invention.
- compound refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function.
- Compounds comprise both known and potential therapeutic compounds.
- a compound can be determined to be therapeutic by testing using the testing methods of the present invention.
- a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
- a compound is said to be "in a form suitable for administration such that the compound is bio-available in the blood of the animal" when the compound may be administered to an animal by any desired route (e.g., oral, intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, etc.) and the compound or its active metabolites appears in the blood of the animal in an active form.
- any desired route e.g., oral, intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, etc.
- anti-neoplastic and “anti-cancer” refer to a compound which arrests or retards the rate of neoplastic progression. The term also refers to a compound which reduces the number of cancer cells in the absence of a change in the rate of neoplastic progression. Anti-neoplastic compounds may be naturally occurring as well as man-made.
- the present invention provides sequences having tissue specific and cell type specific promoter activity.
- the sequences provided herein direct expression to suprabasal cells of stratifying epithelia.
- Also provided by the invention are methods for selectively targeting expression of a gene to a specific tissue and/or a specific cell type at a specific developmental stage within that tissue. These methods provide models for disease as well as for disease therapy and prevention.
- the invention provides transgenic animals in which hINV promoter sequences control the expression of the hINV coding region such that expression is selectively directed to suprabasal cells of certain tissues and not of other tissues.
- the trangenic animals of the invention contain a hINV promoter sequence which directs the expression of human papillomavirus 16 oncogenes. These animals display cervical and epidermal hyperplasias as well as cancer of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung.
- the animals of the invention provide a useful model for screening potential anti-neoplastic compounds, carcinogens, and co-carcinogens for a number of cancers.
- the description of the invention is divided into (a) tissue specific and cell specific promoter sequences, (b) methods for selective gene expression, and (c) uses for the transgenic animals.
- the present invention provides the nucleic acid sequence (SEQ ID NO:l) of a sequence from -2473 to -1 of the hINV gene [the position numbers are designated in relation to the hINV start codon (ATG) in which the adenine is designated as position zero].
- the present invention is not limited to SEQ ID NO:l but specifically contemplates portions thereof. It is preferred that the portions have a length equal to or greater than 10 nucleotides and show greater than 50% homology to SEQ ID NO:l.
- the portion of SEQ ID NO:l is the nucleotide sequence from position -2473 to -7 of Figure 1. In another embodiment, the portion of SEQ ID NO:l is the nucleotide sequence from position -1953 to -7 of Figure 1. In a further preferred embodiment, the portion of SEQ ID NO:l is the nucleotide sequence from position -1333 to -7 of Figure 1. In yet another preferred embodiment, the portion of SEQ ID NO:l is the nucleotide sequence from position -986 to -7 of Figure 1.
- each of the four portions of SEQ ID NO:l was ligated to the hINV coding region (i.e., exon 1, intron 1, and exon 2 which are located in a EcoRI -restricted Charon 4A ⁇ I-3 [Eckert and Green (1986) Cell 46:583-589]) and the resulting constructs used to generate trangenic mice.
- Each of the four portions of SEQ ID NO: 1 directed expression of the heterologous hINV coding sequence to luminal epithelial cells in the kidney.
- the sequence (SEQ ID NO:l) from -2473 to -7 also directed expression of the hINV coding sequence to suprabasal cells of other stratifying tissues, i.e., epidermal, tracheal, and esophageal tissues.
- the hINV upstream sequence from -2473 to -7 was operably linked to the open reading frames of E6 and E7 oncogenes of the HPV- 16 virus.
- the open reading frames of HPV- 16 E6 and E7 are located from the ATG start codon at position 83 to the nucleotide at position 556 which precedes the stop codon for E6, and from the ATG start codon at position 562 to the nucleotide at position 855 which precedes the stop codon for E7 as depicted in Figure 2 (SEQ ID NO:6).
- the resulting hINV-HPV16 construct was used to generate transgenic mice. Data presented herein demonstrates that these transgenic animals expressed E6/E7 in the epidermis and ectocervix, and that in these tissues, expression was selectively directed to suprabasal cells, and not to contiguous basal cells.
- the term "ectocervix” refers to a region of the cervix which is more proximal to the uterus as compared to the "endocervix.” Additionally, the ectocervix is characterized by having stratified squamous epithelial cells whereas the endocervix contains columnar epithelial cells. Furthermore, the epithelial cells of the endocervix are mucus secreting whereas the epithelial cells of the ectocervix are not mucus secreting.
- the sequences of the present invention are not limited to SEQ ID NO:l but include variants of SEQ ID NO:l and portions of these variants.
- variants include, but are not limited to, nucleotide sequences having deletions, insertions or substitutions of different nucleotides or nucleotide analogs. Such variants may be produced using methods well known in the art.
- the present invention is not limited to SEQ ID NO:l but is contemplated to include within its scope homologs of SEQ ID NO:l and portions of these homologs and of variants of these homologs. Homologs which are capable of hybridizing to SEQ ID NO:l and portions thereof may be identified by hybridization at different stringencies.
- the invention provided herein is not limited to SEQ ID NO:l, portions, variants, or homologs thereof having promoter activity, but includes sequences having no promoter activity. This may be desirable, for example, where a fragment of SEQ ID NO:l is used to detect the presence of SEQ ID NO:l or portions thereof in a sample by hybridizing the fragment with nucleic acid sequences in the sample.
- the sequences of the invention are not limited to SEQ ID NO:l, portions, variants, or homologs thereof whose promoter activity is both tissue specific and cell type specific. Rather, sequences having either cell type specific or tissue specific activity are also contemplated to be within the scope of the invention.
- sequences are useful, for example, where it is desirable to target expression of a gene to suprabasal cells in a multiplicity of tissues, or to a multiplicity of tissues without regard to the type of cell targeted.
- portions, variants and homologs of SEQ ID NO:l whose promoter activity is neither cell specific nor tissue specific.
- Such sequences are useful where expression of a gene is desired without regard to either the tissue or cell type in which it is expressed.
- Expression in vitro may be accomplished by operably ligating the gene of interest to sequences of the invention and introducing the ligated expression construct into a cell.
- Expression in vitro may be detected using methods well known in the art, such as detection of the mRNA sequence (e.g., by Northern analysis) and/or of the polypeptide sequence (e.g., by antibody binding) encoded by the gene.
- the present invention is not limited to sense molecules of SEQ ID NO:l but contemplates within its scope antisense molecules comprising a nucleic acid sequence complementary to at least a portion of the polynucleotide of SEQ ID NO:l. These antisense molecules find use in, for example, reducing or preventing expression of a gene whose expression is controlled by SEQ ID NO:l.
- nucleotide sequence of SEQ ID NO:l, portions, variants, homologs and antisese sequences thereof can be synthesized by synthetic chemistry techniques which are commercially available and well known in the art [see Caruthers MH et al., (1980) Nuc.
- fragments of SEQ ID NO:l can be made by treatment of SEQ ID NO:l with restriction enzymes followed by purification of the fragments by gel electrophoresis.
- sequences may also produced using the polymerase chain reaction (PCR) as described by Mullis [U.S. Patent No. 4,683,195] and Mullis et al. [U.S. Patent PCR) as described by Mullis [U.S. Patent No. 4,683,195] and Mullis et al. [U.S. Patent
- nucleotide sequence of synthesized sequences may be confirmed using commercially available kits as well as using methods well known in the art which utilize enzymes such as the Klenow fragment of DNA polymerase I, Sequenase ® , Taq DNA polymerase, or thermostable T7 polymerase. Capillary electrophoresis may also be used to analyze the size and confirm the nucleotide sequence of the products of nucleic acid synthesis, restriction enzyme digestion or PCR amplification. It is readily appreciated by those in the art that the sequences of the present invention may be used in a variety of ways.
- fragments of the sequence of at least about 10 bp, more usually at least about 15 bp, and up to and including the entire (i.e., full-length) sequence can be used as probes for the detection and isolation of complementary DNA sequences. This may be desirable, for example, to determine whether a construct containing sequences of the invention has been integrated into a cell.
- sequences provided herein are also useful in directing the synthesis of polypeptide sequences in vitro and in vivo. This is useful in determining the role of the polypeptide in disease development or treatment, as well as in producing antibodies for diagnostic or therapeutic purposes.
- the present invention provides methods for selectively expressing a nucleotide sequence of interest in a particular cell type and/or a particular tissue. More specifically, the methods provided herein direct expression to stratifying epithelial cells. Yet more specifically, the stratifying epithelial cells are suprabasal cells. In one embodiment, this is accomplished by introducing into an animal cell a vector that contains a nucleotide sequence of interest operably linked to sequences provided herein which have tissue specific and/or cell specific promoter activity. The transfected animal cell is allowed to develop into a transgenic animal in which the nucleotide sequence of interest is expressed in selected cell types and/or tissues. These steps are further described below for specific embodiments. 1. Constructs
- a vector is constructed in which a promoter sequence from -2473 to -7 of Figure 1 is operably linked to a nucleotide sequence of interest.
- the nucleotide sequence of interest is the open reading frame of HPV- 16 E6/E7 oncogenes.
- the nucleotide sequence of interest is the coding region of the hINV gene.
- the invention is not limited to the use of a single portion of the hINV sequence (SEQ ID NO:l) from -2473 and -1 of Figure 1.
- a combination of two or more portions of SEQ ID NO:l are expressly contemplated to be within the scope of the invention.
- a combination of the first and second portions may be desirable to drive expression of a nucleotide sequence of interest in both the first and second tissues.
- SEQ ID NO:l which is tissue specific is a 520 bp sequence located between positions -2473 and -1953.
- Evidence presented herein demonstrates that this 520 bp sequence specifically directs expression of an operably linked sequence to the epidermis and ectocervix.
- nucleic acid sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and of regulatory genes (e.g., activator protein 1 (API), activator protein 2 (AP2), Spl, etc.). Additionally, such nucleic acid sequences include non-coding regulatory elements which do not encode an mRNA or protein product.
- structural genes e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.
- regulatory genes e.g., activator protein 1 (API), activator protein 2 (AP2), Spl, etc.
- nucleic acid sequences include non-coding regulatory elements which do not encode an mRNA or protein product.
- a heterologous promoter which is derived from other than the hINV gene in tandem with promoter sequences of the present invention.
- Such chimeric promoters are included within the scope of the invention and may be desirable where, for example, chimeric promoters result in increased levels of expression of an operably linked downstream coding sequence.
- Chimeric promoters are known in the art and include, for example, the double Tet promoter [Kistner et al. (1996) Proc. Natl. Acad. Sci. USA 93:10933-10938], the UI snRNA promoter-CMV promoter/enhancer [Bartlett et al. (1996) Proc. Natl. Acad. Sci. USA 93:8852- 8857].
- the invention is not limited to nucleotide sequences of interst which comprise a single coding sequence and/or a single non-coding regulatory element.
- a plurality (/ ' . e. , more than one) of coding and non-coding regions which are derived from a plurality of genes may be ligated in tandem such that their expression is controlled by the promoter sequences of the invention.
- a plurality of coding sequences may be desirable, for example, where it is useful to express a transcription product of more than one gene to permit interaction of these transcriptional products.
- the open reading frames (ORFs) of the E6 oncogne and E7 oncogne of HPV- 16 are ligated such that their expression is controlled by an hINV promoter sequence of the invention.
- E6 and E7 ORF sequences may be modified by previously described methods [e.g., Sambrook et al., (1989) supra; Methods in Enzymology (1987) Vol. 152, Guide to Molecular Cloning Techniques (Berger and Kimmerl (Eds.), San Diego: Academic Press, Inc.]
- a plurality of coding sequences may be desirable where one of the gene sequences is a reporter gene sequence.
- reporter gene sequences include the sequences encoding the enzymes ⁇ -galactosidase and luciferase.
- Fusion genes may also be desirable to facilitate purification of the expressed protein.
- the heterologous sequence which encodes protein A allows purification of the fusion protein on immobilized immunoglobulin.
- Other affinity traps are well known in the art and can be utilized to advantage in purifying the expressed fusion protein.
- pGEX vectors Promega, Madison WI
- GST glutathione S-transferase
- such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
- fusion polypeptides useful in the purification of the coiled coil polypeptide are commercially available, including histidine tails (which bind to Ni 2+ ), biotin (which binds to streptavidin), and maltose-binding protein (MBP) (which binds to amylose). Proteins made in such systems are designed to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released at will from the heterologous polypeptide moiety to which it is fused.
- histidine tails which bind to Ni 2+
- biotin which binds to streptavidin
- MBP maltose-binding protein
- nucleic acid sequences of interest may be either contiguous or separated by intervenimng polynucleotide sequences, so long as the nucleic acid sequences of interest are placed in- frame.
- Expression vectors in which expression of a nucleic acid sequences of interest is controlled by promoter sequences of the invention may be constructed using techniques well known in the art. [Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold
- nucleic acid sequences of interest is placed in operable combination with the hINV promoter sequences of the invention in the presence of transcription and translation regulatory sequences.
- Regulatory sequences include initiation signals such as a start codon (i.e., ATG), enhancers, and transcription termination signals.
- ATG start codon
- enhancers enhancers
- transcription termination signals The ATG initiation codon must be in the correct reading frame to ensure translation of the entire heterologous nucleotide sequence.
- Transcription termination signals are placed downstream of the heterologous nucleic acid sequence and include polyadenylation sequences which are exmplified by, but not limited to, SV40 poly-A sequence, hINV poly-A sequence, or bovine growth hormone poly-A sequence, etc.
- the initiation signals are those of the heterologous nucleotide sequence.
- the polyadenylation signal of SV40 is used.
- RNA stability may affect RNA stability as well as enhancers (i.e., a sequence which when activated resutls in an increase in the basal rate of transcription of a gene) and silencers (i.e., a sequence involved in reducing expression of a gene) may also be included.
- enhancers i.e., a sequence which when activated resutls in an increase in the basal rate of transcription of a gene
- silencers i.e., a sequence involved in reducing expression of a gene
- These regulatory sequences may be relatively position-insensitive, /. e. , the regulatory element will function correctly even if positioned differently in relation to the heterologous nucleotide sequence in the construct as compared to its position in relation to the corresponding heterologous nucleotide sequence in the genome.
- an enhancer may be located at a different distance from the hINV promoter sequence, in a different orientation, and/or in a different linear order.
- regulatory sequences in constructs which contain hINV upstream sequences of the invention.
- Any number, type and location of regulatory sequences may be used with the sequences of the present invention provided that such regulatory sequences do no substantially interfere with the desired activity (e.g., promoter activity, tissue specific promoter activity, cell type specific promoter activity, ability to hybridize to homologous nucleotide sequences, etc.) of the sequences of the invention.
- the expression vector which contains the hINV promoter sequences of the invention in operable combination with a nucleic acid sequences of interest is transfected into a host cell.
- Host cells include bacterial, yeast, plant, insect, and mammalian cells.
- the host cell is mammalian.
- the host cell is a mouse cell.
- Any number of selection systems may be used to recover transfected cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22:817-23) genes which can be employed in tk “ or aprt " cells, respectively.
- antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate [Wigler M et al, (1980) Proc Natl Acad Sci 77:3567-70]; npt, which confers resistance to the aminoglycosides neomycin and G-418 [Colbere-Garapin F et al, (1981) J. Moi. Biol. 150:1-14] and als o ⁇ pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra).
- trpB which allows cells to utilize indole in place of tryptophan
- hisD which allows cells to utilize histinol in place of histidine
- FISH Fluorescent In Situ Hybridization
- DNA or RNA can be isolated from cells for detection of the transgene by Southern or Northern hybridization or by amplification based assays.
- Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on sequence of the nucleic acid sequence of interest in order to detect cells and tissues which contain the DNA or RNA encoding the transgene of interest.
- oligonucleotides and “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.
- Standard PCR methods useful int he present invention are described by Innis et al. (Eds.), "PCR Protocols: A Guide to Methods and Applications,” Academic Press, San Diego (1990)].
- heterologous nucleic acid sequences are by detecting the polypeptide product of transcription of the heterologous nucleotide sequence.
- a variety of protocols which employ polyclonal or monoclonal antibodies specific for the protein product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescent activated cell sorting
- a competitive binding assay may also be used.
- a two-site, monoclonal-based immunoassay which utilizes monoclonal antibodies that are reactive to two non-interfering epitopes on the protein of interest may be employed. These and other assays are described in, among other places, Hampton R et al.
- RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
- RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
- Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
- the present invention provides a number of transgenic animals.
- the transgenic animals of the present invention provide the first animal model of colon cancer.
- a first transgenic animal model of anal cancer is also provided.
- a transgenic animal model of cervical and epithelial hyperplasia is provided.
- transgenic animals are provided in which expression of any nucleic acid sequences of interest is selectively targeted to luminal epithelial cells of the kidney in the presence/absence of expression in suprabasal eels of the epidermis, cervix, etc.
- These animals provide useful models for the identification of potential carcinogens and co-carcinogens, identification of anti-neoplastic compounds, identification of genes which play a role in neoplastic progression of cancers of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen, lung, and cervix.
- a construct i.e., hINV-HPV16
- hINV-HPV16 human papillomavirus 16
- HPV- 16 human papillomavirus 16
- This construct was used to generate trangenic mice which expressed E6 and E7 mRNA in a tissue-specific and differentiation appropriate manner.
- full-length and spliced E6 and E7 mRNA was expressed in stratifying epithelial tissue, such as the skin, cervix and urothelial lining.
- E6 and E7 mRNA expression was localized in suprabasal cells and not in the less differentiated contiguous basal cells of the same tissue.
- Transgenic mice which are heterozygous for the hINV-HPV16 construct developed neoplasias of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung, as well as epidermal and ectocervical hyperplasias by the age of 7 months.
- a first step in the generation of the transgenic animals of the invention is the introduction of a construct containing the desired heterologous nucleic acid sequence under the expression control of hINV upstream sequences of the invention into target cells.
- Several methods are available for introducing the expression vector which contains the heterologous nucleic acid sequence into a target cell, including microinjection, retroviral infection, and implantation of embryonic stem cells. These methods are discussed as follows.
- the cell is implanted into the uterus of a pseudopregnant female and allowed to develop into an animal.
- 70% carry the expression vector sequence in all of their cells, including the germ cells.
- the remaining 30% of the transgenic animals are chimeric in somatic and germ cells because integration of the expression vector sequence occurs after one or more rounds of replication.
- Heterozygous and homozygous animals can then be produced by interbreeding founder transgenics. This method has been successful in producing transgenic mice, sheep, pigs, rabbits and cattle [Jaenisch (1988) supra; Hammer et al., (1986) J. Animal Sci.:63:269; Hammer et al., (1985) Nature 315:680-683; Wagner et al.,
- Retroviral infection of preimplantation embryos with genetically engineered retroviruses may also be used to introduce transgenes into an animal cell.
- blastomeres have been used as targets for retroviral infection [Jaenisch, (1976) Proc. Natl. Acad. Sci USA 73:1260-1264].
- Transfection is typically achieved using a replication- defective retrovirus carrying the transgene [Jahner et al, (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten et al, (1985) Proc. Natl. Acad Sci USA 82:6148-6152].
- Transfection is obtained, for example, by culturing eight-cell embryos, from which the zona pellucida has been removed with fibroblasts which produce the virus [Van der Putten (1985), supra; Stewart et al, (1987) EMBO J. 6:383-388]. The transfected embryos are then transferred to foster mothers for continued development. Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele [Jahner et al, (1982) Nature 298:623-628]. Yet another alternative method involves intrauterine retroviral infection of the midgestation embryos [Jahner et al. (1982), supra ⁇ .
- retroviral infection methods include the ease of transfection and the insertion of a single copy of the transgene, which is flanked by the retroviral long terminal repeats (LTRs), into the chromosome.
- LTRs retroviral long terminal repeats
- this method is not a preferred method because most of the founders will show mosaicism since infection occurs after cell division has begun. This necessitates outbreeding to establish homozygous and heterozygous lines suitable for analysis of gene expression.
- the retroviral LTR sequences may interfere with the activity of the hINV upstream sequences in directing expression of the heterologous nucleic aid sequences.
- ES cells embryonic stem cells
- ES cells are pluripotent cells directly derived from the inner cell mass of blastocysts [Evans et al, (1981) Nature 292:154-156; Martin (1981) Proc. Natl. Acad Sci. USA 78:7634-7638; Magnuson et al, (1982) J. Embryo. Exp. Morph. 81:211-217; Doetchman et al, (1988) Dev. Biol. 127:224-227], from inner cell masses [Tokunaga et al, (1989) Jpn. J.
- Expression vectors can be introduced into ES cells using any method which is suitable for gene transfer into cells, e.g., by transfection, cell fusion, electroporation, microinjection, DNA viruses, and RNA viruses [Johnson et al, (1989) Fetal Ther. 4 (Suppl. l):28-39].
- ES cells are the most pluripotent cultured animal cells known. For example, when ES cells are injected into an intact blastocyst cavity or under the zona pellucida, at the morula stage embryo, ES cells are capable of contributing to all somatic tissues including the germ line in the resulting chimeras. Once the expression vector has been introduced into an ES cell, the modified ES cell is then introduced back into the embryonic environment for expression and subsequent transmission to progeny animals.
- ES cells The most commonly used method is the injection of several ES cells into the blastocoel cavity of intact blastocysts [Bradley et al, (1984) Nature 309:225-256].
- a clump of ES cells may be sandwiched between two eight-cell embryos [Bradley et al, (1987) in "Teratocarcinomas and Embryonic Stem Cells: A Practical
- RNA probe which is specific for a portion of (or the entire) gene of interest, e.g., by using PCR.
- the PCR amplified fragment is subcloned into a plasmid (e.g., pBluescript (Stratagene)) and the RNA probe synthesized using labelled UTP (e.g., 35 S-UTP) and RNA polymerase (e.g., T3 or T7 polymerase (Promega)).
- Paraffin-embedded tissue sections are mounted on slides, deparaffinized, rehydrated and the protein digested (e.g., with proteinase K), then dehydrated prior to hybridization with the RNA probe at the desired hybridization stringency. Slides are then developed for autoradiography using commercially available developers. Labelling of tissues and cells as detected on the autoradiographs indicates expression in those tissues and cells of the mRNA encoded by the gene of interest. Alternatively, mRNA encoded by the gene of interest may be detected by reverse transcription polymerase chain reaction (RT-PCR) as described herein (see, e.g., Example 3).
- RT-PCR reverse transcription polymerase chain reaction
- expression of the protein product of the gene of interest may be determined using immunohistochemical techniques. Briefly, paraffin-embedded tissue sections are dewaxed, rehydrated, treated with a first antibody which is specific for the polypeptide product of the gene of interest. Binding is visualized, for example, by using a secondary biotinylated antibody which is specific for the constant region of the primary antibody, together with immunoperoxidase and 3,3'-amiobenzidine as a substrate. Sections may then be stained with hematoxylin to visualize the cellular histology. Antibody binding of tissues and cells which is detected by antibody binding demonstrates expression of the protein product of the gene of interest in these tissues and cells.
- Yet another alternative method for the detection of expression of the protein product of the gene of interest is by Western blot analysis wherein protein extracts from different tissues are blotted onto nitrocellulose filters, and the filters incubated with antibody against the protein product of the gene of interest, followed by detection of antibody binding using any of a number of available labels and detection techniques (see, e.g., Example 3).
- the transgenic animals of this invention may be used to (a) screen compounds for anti-neoplastic activity, (b) screen compounds for carcinogenic and co-carcinogenic activity, (c) identify genes which play a role in neoplastic progression of tracheal, easophageal, colon, epidermal, anal/rectal, lymph node, spleen, lung and cervical cancers, and (d) provide an in vivo model for tracheal, easophageal, colon, epidermal, anal/rectal, lymph node, spleen, lung and cervical cancers.
- transgenic animals In using the transgenic animals provided herein to screen potential anti-neoplastic compounds, it is anticipated that presently used compounds (e.g., the retinoids which have already been tested in clinical trials in patients with HPV disease) and anti-cancer compounds currently in use for chemotherapy of cancers of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung, in humans will be screened first because many of their effects on humans are already known.
- compounds e.g., the retinoids which have already been tested in clinical trials in patients with HPV disease
- anti-cancer compounds currently in use for chemotherapy of cancers of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung
- the screening process can be used to gather data such as which compounds are most effective at particular stages of tracheal, easophageal, colon, epidermal, anal/rectal, lymph node, spleen, lung, and ectocervical cancer development.
- compounds which are derivatives of existing efficacious anti-cancer agents, or which have a new mechanism of action may also be administered singly or in combination to determine their effect in altering the incidence, rate of development, or pathology of cancers of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen, lung, and ectocervix.
- transgenic mice of this invention Another use of the transgenic mice of this invention is to screen potential carcinogens and co-carcinogens.
- One of skill in the art would appreciate that this may be achieved by exposing transgenic animals of this invention, which exhibit pre-neoplastic lesions (e.g., hyperplasias and dysplasias) to agents which -ire suspected of having carcinogenic or co- carcinogenic activity. These agents are administered either singly or in combination. Where a combination of agents is used, the agents may be administered simultaneously or sequentially.
- trangenic animals An additional use of the trangenic animals provided herein is to determine the identity of genes which are involved in the cellular progression to pre-neoplastic and neoplastic states in epithelial tissues. This may be done, for example, by mating two different transgenic mice (e.g., a transgenic mouse which contains a gene or oncogene whose expression is under the control of a hINV promoter sequence, and another transgenic mouse containing HPV oncogenes that are regulated by a hINV promoter sequence) to produce a double transgenic animal. The double trangenic animal is then used to determine the frequency and rate of development of pre-neoplastic and neoplastic lesions.
- transgenic mice e.g., a transgenic mouse which contains a gene or oncogene whose expression is under the control of a hINV promoter sequence, and another transgenic mouse containing HPV oncogenes that are regulated by a hINV promoter sequence
- genes or oncogenes which accelerate malignant progression in tracheas, easophageal, colon, epidermal, anal/rectal, lymph node, spleen, lung, as well as ectocervical tissues, or which induce tumors in other than these tissues provides further targets for therapeutic treatment.
- Treatment may be accomplished, for example, by administering to the animal of anti-sense nucleotide sequences which target the coding or non-coding regions of these gene and oncogenes, and/or of antibodies against the polypeptide products of the genes or oncogenes which are identified to play a role in malignant progression.
- a further use of the herein provided transgenic animals is to develop an in vivo model for cervico-vaginal neoplastic progression.
- Human papillomaviurses are believed to be the etiologic agents for the majority of human cervical carcinoma. It is also believed that the HPV- 16 E6 and E7 oncogenes, as well as sex hormones, play a significant role in the development of cervical cancer.
- the involvement of estrogen, estrogen-like compounds, and estrogen agonists and antagonists e.g. tamoxifen and megestrerol
- This model would then provide a system to screen candidate drugs (as described supra) (e.g., anti- estrogens and progestins) for their ability to circumvent cervico-vaginal neoplastic progression in this model.
- transgenic animals involve administration of potential anti-neoplastic compounds, carcinogens, or co- carcinogens alone or in combination, as well as the detection of the effect of such administration on cancer development and/or progression.
- Administration of potential anti- neoplastic compounds, carcinogens, co-carcinogens, and other compounds of interest is accomplished using any suitable route (e.g., oral, parenteral, rectal, controlled release transdermal patches and implants, etc.).
- Methods of parenteral delivery include topical, intra-arterial (e.g., directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
- routes of administration will depend on the stability of the compound, the susceptibility of the compound to "first pass" metabolism, the concentration needed to achieve a therapeutic effect, and the like.
- these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Maack Publishing Co, Easton PA).
- detection of the effect of the compound being tested on cancer may be determined according to standard techniques well-known in the art. These techniques include visual inspection, immunohistochemical techniques, and the like. For example, the change in the size of tumors may be monitored using calipers. The relative number and distribution of hyperplastic and dysplastic cells in relation to normal cells may be determined by histochemical analysis in combination with incorporation of 5-bromo- 2'-deoxyuridine (BrdU) incorporation. Briefly, animals are injected intraperitoneally with 100 ⁇ g/g body weight of a 5 mg/ml solution of BrdU (Sigma) in a 10 mM Tris, 0.9% saline, 1 mM EDTA pH 8.0 buffer.
- BrdU 5-bromo- 2'-deoxyuridine
- Antibody binding is detected using a peroxidase/avidin/biotin complex (ABC) (Vector Laboratories) with 3,3'- diaminobenzidine (Sigma) as the chromogen. Dividing cells (i.e., which incorporate BrdU) are then visualized using microscopy.
- ABSC peroxidase/avidin/biotin complex
- a compound that appears promising is further evaluated by administering various concentrations of the compound to the transgenic animals provided herein in order to determine an approximate therapeutic dosing range.
- Animal testing may be supplemented and confirmed by testing on human subjects.
- animal models herein provided allow the testing of a large number of compounds, both by the methods described above and other methods known in the art, in a system similar in many important respects to that in humans.
- the nucleic acid sequence of the upstream promoter region of the human involucrin (hINV) gene was determined. Involucrin is expressed specifically in the suprabasal layers of the human epidermis. Factors affecting the regulation of hINV gene expression and partial sequences located upstream of the hINV gene have been reported [Carroll et al. (1992) J. Cell Sci. 103:925-930; Takahashi et al. (1993) J. Investi. Dermatol. 100:10-15; Choo et al. (1993) Exper. Cell Res. 208:161-169;
- Plasmid pSP64 ⁇ I-3 H6B [Eckert et al., (1986) Cell 46:583-589] was restricted with Hindlll and Kpnl, and the released DNA fragment was subcloned into Hindlll/KpnI-restricted pGEM7Zf(+) (Promega). The resulting DNA product was transfected into HB101 bacteria and an insert-containing plasmid clone, pGEM7Z ⁇ I-3 H/K, was isolated using standard methods [Maniatis et al., (1982) "Molecular Cloning - A Laboratory Manual," Cold Spring
- the region from the Kpnl site (position -986) to the Celll site at position -1 was sequenced by cloning a KpnI/XhoI piece of the gene (this segment includes bases -1 to the Kpnl site at -986 ( Figure 1) and extends several hundred bases downstream beyond Celll to the Xhol site.
- This segment was subcloned into KpnI/XhoI-restricted plasmid pBluescript II KS +/- (Stratagene), the resulting DNA product was transfected into HB101 bacteria, and an insert-containing plasmid clone, pGEM7Z ⁇ I-3 H/K, was isolated using standard methods (Maniatis et al. (1982) supra). The insert in this plasmid was sequenced. The ends of this fragment were sequenced using the T3 and T7 universal promoter primers (Promega) and the
- the sequence (SEQ ID NO:l) of the sense strand of the hINV upstream sequence between positions -2473 and -1 is shown in Figure 1.
- the sequence of the sense strand was complementary to the sequence of the anti-sense strand. This complementarity confirmed the fidelity of the sequencing method used.
- the location of selected transcription factor binding sites is indicated in Figure 1.
- the activator protein 1 (API) sites i.e., AP1-1, -2, -3, -4 and -
- Transgenic mice which harbor and express HPV- 16 E6 and E7 oncogenes under the transcriptional control of a hINV promoter sequence from -2473 to -7 were produced by introducing an expression construct into a one cell embryos of a mouse, allowing further development of the mouse in a pseudopregnant female, selecting offspring which contained the transgene, and determining the phenotype of the offspring. These steps are described in more detail below.
- the upstream region of the hINV gene was isolated by digesting pSP64 ⁇ I-3 H6B (Eckert et al. (1986) supra) to completion with EcoRI (EcoRI, and all other restriction enzymes, were obtained from New England Biolabs, Beverly, MA), followed by a partial digestion with Hindi. This released an
- the resulting insert was transferred to Smal-digested pUC1813 (Kay and McPherson, Nucl Acids Res (1987) 15:2778) to yield plasmid pUC1813(-2473/-7).
- This plasmid was in turn digested with BamHI and the insert was subcloned into Bglll-restricted and alkaline phosphatase (Boehringer Mannheim)-phosphatased pGL2-basic (Promega). This yielded pINV-2473.
- pINV-2473 was digested with Hindlll/BamHI and the insert was discarded. The remaining plasmid intermediate, designated *, was saved for use as outlined in the next paragraph.
- HPV 16 E6/E7 genes For the preparation of the HPV 16 E6/E7 genes, the HPV segment encoding E6/E7 was derived from plasmid pUC19 HPV16 E6/E7-SV40 BamH1.
- pUC19 HPV16 E6/E7- SV40 BamHI. was prepared by simultaneously digesting pUC8-HPV16, which contains the complete 8 kilobase HPV16 genome sequence described by Seedorf et al. (1985) Virology 145:181-185 and shown in Figure 2 (SEQ ID NO:6) cloned into the BamHI site in pUC8 (Gibco-BRL), with Kpnl and EcoRI.
- the released 1330 bp fragment containing the Ex/E7 coding region of HPV 16 was subcloned into EcoRI/KpnI-restricted pUC19 (Gibco-BRL) to yield plasmid pUC19 HPV 16 (E 7453 /K g80 ),.
- This plasmid was digested with Hindlll/BamHI and the insert, which contains the HPV 16 E6/E7 genes and the SV40 termination sequences, was mixed with * and ligated with T4 polynucleotide kinase (Boehringer-Mannheim).
- the DNA products were transfected into HB101 bacteria, and an insert-containing plasmid clone, pINV-2473(E6/E7) was isolated using standard methods (Maniatis et al. (1982) supra).
- the ⁇ INV-2473(E6/E7) plasmid was restricted with Nhel/Bam ll to release the eukaryotic expression segment of the plasmid, i.e., hINV-E6/E7-SV40 STOP .
- Fertilized single cell embryos from a C57BL/6 (Charles River) x SJL (Charles River) mating were microinjected with the hINV-E6/E7-SV40 STOP DNA fragment using standard methods [Hogan et al. (1986) "Manipulating the mouse embryo: A laboratory manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] and implanted into recipient pseudo-pregnant C57BL/6 females (Charles River).
- the offspring were characterized for the presence of the E6/E7 oncogenes by PCR analysis and Southern blotting of tail DNA using previously described methods [Falcinelli et al. (1992) J Med. Virol 37:93-98; Sambrook et al., (1989) Molecular Cloning, a laboratory manual (2 nd Ed), Cold Spring Harbor Lab, Cold Spring, NY]. Trangenic mice which were homozygous for the hINV-2473(E6/E7) transgene were produced by mating mice which were heterozygous for this transgene.
- Figure 4 shows a Southern blot of tail DNA from transgenic offspring which were heterozygous for the E6/E7 oncogenes. Southern blots were probed with plasmid pINV-2473
- E6/E7 which was made radioactive by random priming in the presence of ⁇ "32 P-dCTP.
- Transgenic offspring heterozygous for the E6/E7 oncogenes were identified by the presence of a 1.7 kb band on the gel, that was absent from control offspring which developed from C57BL/6 x SJL embryos.
- the Southern blots show that the transgenic offspring contained E6/E7 DNA.
- E6 E7 RNA production was detected using PCR amplification of RNA isolated from the epidermis of transgenic E6/E7 heterozygous mouse.
- the primers used for PCR amplification were 5'-GTG TGT ACT GCA AGC AAC AG (upstream primer) (SEQ ID NO:2) and 5'-GCA ATG TAG GTG TAT CTC CA (downstream primer) (SEQ ID NO:3). These sequences were homologous to the HPV16 E6 gene coding region.
- RNA was isolated using standard methods (Maniatis et al., 1982).
- the products of the reverse transcription reaction were added to a PCR reaction which contained 3 units of Tag DNA polymerase (Boehringer Mannheim, Indianapolis), 10 ⁇ M of each PCR primer, 100 ⁇ M of deoxynucleotides in a buffer of 100 mM Tris-HCl, pH 8.3, 15 mM MgCl 2 , and 500 mM KC1.
- PCR was performed for 30 cycles. Each cycle was 94 C for 2 min, 45 C for 2.5 min, and 72 C for 2.5 min.
- the possible products of this amplification that could be detected include bands of 395, 213 and 95 bp. Any combination of these bands may be detected depending upon the pattern by which the HPV E6 region RNA is spliced. Bands of 395 and 213 bp were detected (data not shown), thus demonstrating that HPV 16 RNA was produced in the transgenic mice.
- transgenic HPV 16 E6/E7 animals Of the 10 lines of transgenic HPV 16 E6/E7 animals, 7 displayed hair loss and/or reduced body size and/or tumor formation. Hair loss was observed on the face, underside and back. Trangenic animals also were of 10%-20% reduced size compared to control wild-type mice. Importantly, both heterozygous and homozygous transgenic HPV 16 E6/E7 mice developed palpable tumors of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung, as well as epidermal and ectocervical hyperplasias (Table 2). Epidermal scarring was observed on the underside and on the ears. The severity of the phenotype and the subgroup of tumors that developed varied with each mouse line and was generally more severe in homozygotes as compared to heterozygotes.
- Heterozygous HPV 16 E6/E7 transgenic mice displayed epidermal and ectocervical hyperplasia, as well as carcinoma of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung.
- DMEM Dulbecco's modified Eagle's medium
- GibcoBRL Dulbecco's modified Eagle's medium
- GibcoBRL fetal calf serum
- Cells were cultured on these dishes until confluent and were then transferred to standard tissue culture plates (Falcon 3003) (Becton Dickinson Labware).
- the cells were passaged by treating confluent cultures in 50 cm 2 surface area dishes with 5 ml of trypsin at the manufacturer's (GibcoBRL) recommended concentration for 10 min. This released the cells from the surface.
- the released cells were then split at a ratio of 1:10 and plated into Falcon 3003 dishes, grown until confluent and then passaged onto additional Falcon 3003 dishes.
- the resulting cell lines could be maintained indefinitely in this manner.
- Aliquots of these cells were cryogenically frozen at between passages 15 and 20 by resuspension of approximately 1 million cells in tissue culture medium (DMEM) containing 10% fetal calf serum supplemented to contain 15% glycerol and slow cooling in a -70 C freezer. Once frozen, the cells were transferred to liquid nitrogen for storage.
- tissue culture medium DMEM
- constructs containing different hINV upstream regions ligated to hINV coding sequences were used to generate transgenic mice and the transgenic animals screened to tissue-specific and cell-specific expression of hINV as described below.
- E16E, H6B, Ha5.5B, A4.3B, and K4B Five expression plasmids (i.e., E16E, H6B, Ha5.5B, A4.3B, and K4B) were constructed to contain the hINV coding region (i.e., exon 1, intron 1, and exon 2) under the regulatory control of different hINV upstream sequences.
- the structure of the deletion hINV constructs is shown in Figure 5. Briefly, the E16E construct contained a 8 kb hINV upstream sequence located between the EcoRI recognition site upstream of the hINV start codon, the transcribed region of the hINV gene and 5.0 kb of hINV sequence downstream of the hINV stop codon.
- the remaining four constructs were deletion constructs which were obtained by truncating the E16E construct at a BamHI site located just downstream of the transcribed hINV gene and by progressively deleting sequences at the 5' end of the E16E construct.
- the H6B, Ha5.5B, A4.3B, and K4B constructs represented progressively truncated forms of the E16E construct in which progressively longer fragments at the 5' of the BamHl- truncated E16E were deleted to leave 2,473 bp, 1953 bp, 1333 bp, and 986 bp of the hINV upstream region, respectively (Figure 5).
- the top line indicates the distance in basepairs.
- the extent of each transgene is indicated by the length of the thin lines.
- the vertical line and black box indicates the first and second exons, respectively.
- the arrow indicates the transcription start site and direction of transcription.
- E16E was constructed by EcoRI restriction of Charon 4A ⁇ I-3 (Eckert and Green, Cell
- the EcoRI fragment was then purified and subcloned into pBKS(+) (Stratagene, San Diego, CA) to yield pBKS E16E.
- the EcoRI insert from this plasmid, which was injected to make the E16E mice is shown in Figure 5.
- the H6B transgene was a 6 kb Hindlll/BamHI fragment containing the complete hINV gene that was derived by cloning Charon 4ALI-3 with Hindlll/BamHI and subcloning into Hindlll/BamHI-restricted pSP64 (Eckert et al, (1986) Cell 46:583-589).
- the deletion constructs were restricted with BamHI and either EcoRI (for ⁇ 16 ⁇ ), Hindlll (for H6B), H ell (for ⁇ a5.5B), Accl (for A4.3B) or Kpnl (for K4B) to release the eukaryotic expression segment of the plasmids. Fertilized single cell embryos from a C57BL/6 x SJL mating were microinjected separately with the released DNA fragments as described supra, and implanted into recipient pseudo-pregnant C57BL/6 females.
- the offspring were characterized for the presence of each transgene by Southern blotting of tail DNA as described supra, using probes that corresponded exactly to the microinjected transgene. Southern blot analysis showed that one or more heterozygous transgenic mouse lines which contained the hINV transgene were generated by microinjection of each of the five constructs (data no shown). Homozygous transgenic lines for each transgene were produced by mating mice which were heterozygous for that transgene.
- Figure 6 shows the results of a Western blot of hINV protein expression in epidermis. Extracts from epidermis from E16E and H6B mouse lines and a non-transgenic line (NT) were electrophoresed on a gel and western blotted using an hINV-specific antibody as previously described [Crish et al. 91993) Differentiation 53:191-200]. Briefly, total protein extracts (approximately 1 mg/ml) were prepared in Laemmli sample buffer and equivalent quantities (20 ⁇ g/lane) were electrophoresed on an 8.5% acrylamide gel and transferred to nitrocellulose. Involucrin immunoreactive material was detected by incubating with an involucrin-specific antibody [Rice et al.
- mice transgenic for the Ha5.5B, A4.3B or K4B constructs expressed hINV only in the kidney, and not in the epidermis or cervix.
- Non-transgenic (NT) littermates did not produce hINV.
- the observed selective decrease in transgene expression in stratifying tissues other than the kidney, as compared to transgene expression in the kidney is unlikely to be the result of the removal of a general enhancer, since loss of such an enhancer would uniformly reduce transgene expression in all tissues, in contrast to the observed consistent selective loss of expression from non-kidney tissue.
- this data demonstrates that the nucleic acid sequence (SEQ ID NO:l) ( Figure 1) which is located between positions -986 bp and -7 bp upstream of the hINV gene is necessary for transgene expression in the kidney.
- RT-PCR reverse transcription polymerase chain reaction
- the hINV sense primer was 5' -CTC CAC CAA AGC CTC TGC-3' (SEQ ID NO:4) beginning at position +2477 in the untranslated region of exon 1 of the hINV gene; the hINV reverse primer was 5'-CTG CTT AAG CTG CTG CTC-3' (SEQ ID NO:5) wherein the 5'-end of this primer was positioned at position +4063 of exon of the hINV gene.
- Amplification was carried out in a Perkin-Elmer Thermal Cycler using the following cycling protocol: 2 min. at 94°C; 2 min. 30 sec. at 55°C; 2 min. 30 sec. at 72°C.
- Immunohistological analysis was carried out in order to evaluate whether the localization of the observed tissue specific expression of transgenes controlled by hINV upstream sequences was differentiation appropriate, i.e., targeted to suprabasal layers of stratifying squamous epithelia.
- Epidermal and cervical tissues were fixed in buffered formalin, embedded in paraffin and sectioned using methods well known in the art. The sections were deparaffinized, blocked, incubated with primary anti-hlNV antibody (Rice et al. (1979) supra) at a dilution of 1:1000 and secondary detection agents as previously described
- hINV protein was detected in the upper spinous and granular layers of the epidermis (i.e., footpad) in E16E and H6B transgenic mice. Suprabasal expression was also observed in the ectocervical epithelium in E16E and H6B transgenic mice, in the absence of basal cell staining. As expected from the Western blots described above, no expression was observed in the ectocervix or epidermis of transgenic Ha5.5B, A4.3B and K4B mice.
- mice which were transgenic for E16E or H6B expressed the respective transgenes in the stratifying squamous epithelial tissue of the cervix and epidermis were transgenic for E16E or H6B expressed the respective transgenes in the stratifying squamous epithelial tissue of the cervix and epidermis.
- the present invention provides nucleic acid sequences which are useful for directing expression of a nucleotide sequence of interest to a specific cell type in a specific tissue at a specific developmental stage of the cell.
- the sequences provided herein are capable of directing expression in suprabasal cells of stratifying squamous epithelial tissue and in uroepithelial tissue.
- the present invention provides transgenic animals which provide a unique model for colon cancer and anal/rectal cancers.
- animal models are also useful for screening potential anti- neoplastic compounds, carcinogens, and co-carcinogens for cancers of the trachea, esophagus, colon, epidermis, anus/rectum, lymph node, spleen, lung, and cervix.
- MOLECULE TYPE DNA (genomic)
- GCATGATCCA GGAACATGAC AAAATTAGTG CTTTAAATAG ATTTACTTGG GCTAAGAGAA 780
- TAATAGTCAT TATTTTCGGG TCGTAAAATT CACTGTTCTT CAATGAATAC TTGTAGAGCA 1080
- CTATCTTCAA ACATATAACC CAGCCTGGAC ATCCCCGAAA GACACATAAC TTTCCATTTC 2100
- MOLECULE TYPE other nucleic acid
- MOLECULE TYPE other nucleic acid
- MOLECULE TYPE other nucleic acid
- MOLECULE TYPE other nucleic acid
- MOLECULE TYPE DNA (genomic)
- GACAGTGCTC CAATCCTCAC TGCATTTAAC AGCTCACACA AAGGACGGAT TAACTGTAAT 3600
- AGTACCTACG ACATGGGGAG GAATATGATT TACAGTTTAT TTTTCAACTG TGCAAAATAA 6780
- AAAAATACAC TTTTTGGGAA GTAAATTTAA AGGAAAAGTT TTCTGCAGAC CTAGATCAGT 7020
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JP54595598A JP2001517956A (en) | 1997-03-24 | 1998-03-24 | Tissue-specific promoter |
AU68696/98A AU6869698A (en) | 1997-03-24 | 1998-03-24 | Tissue specific promoters |
CA002285413A CA2285413A1 (en) | 1997-03-24 | 1998-03-24 | Tissue specific promoters |
EP98914305A EP0972013A4 (en) | 1997-03-24 | 1998-03-24 | Tissue specific promoters |
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PCT/US1998/005866 WO1998042826A1 (en) | 1997-03-24 | 1998-03-24 | Tissue specific promoters |
PCT/US1998/005588 WO1998042825A1 (en) | 1997-03-24 | 1998-03-24 | A transgenic model of colon cancer and anal cancer |
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US5578444A (en) * | 1991-06-27 | 1996-11-26 | Genelabs Technologies, Inc. | Sequence-directed DNA-binding molecules compositions and methods |
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US5698764A (en) * | 1994-06-09 | 1997-12-16 | The Regents Of The University Of California | Transgenic mice expressing HPV early region oncogene develop progressive epithelial neoplasia |
US5709844A (en) * | 1994-06-09 | 1998-01-20 | The Regents Of The University Of California | Transgenic mice expressing HPV early region oncogene develop progressive cervico-vaginal neoplasia |
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1998
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- 1998-03-24 CA CA002284735A patent/CA2284735A1/en not_active Abandoned
- 1998-03-24 JP JP54595598A patent/JP2001517956A/en active Pending
- 1998-03-24 WO PCT/US1998/005588 patent/WO1998042825A1/en active Application Filing
- 1998-03-24 AU AU67677/98A patent/AU6767798A/en not_active Abandoned
- 1998-03-24 EP EP98914305A patent/EP0972013A4/en not_active Withdrawn
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Non-Patent Citations (6)
Title |
---|
CARROLL J. M., TAICHMAN L. B.: "CHARACTERIZATION OF THE HUMAN INVOLUCRIN PROMOTER A TRANSIENT BETA-GALACTOSIDASE ASSAY.", JOURNAL OF CELL SCIENCE, CAMBRIDGE UNIVERSITY PRESS, LONDON, GB, vol. 103., 1 January 1992 (1992-01-01), GB, pages 925 - 930., XP002912489, ISSN: 0021-9533 * |
CRISH J. F., ET AL.: "TISSUE-SPECIFIC AND DIFFERENTIATION-APPROPRIATE EXPRESSION OF THE HUMAN INVOLUCRIN GENE IN TRANSGENIC MICE: AN ABNORMAL EPIDERMA PHENOTYPE.", DIFFERENTIATION., SPRINGER VERLAG., DE, vol. 53., 1 January 1993 (1993-01-01), DE, pages 191 - 200., XP002912485, ISSN: 0301-4681 * |
LOPEZ-BAYGHEN E., ET AL.: "TRANSCRIPTIONAL ANALYSIS OF THE 5'-NONCODING REGION OF THE HUMAN INVOLUCRIN GENE*.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 271., no. 01., 5 January 1996 (1996-01-05), US, pages 512 - 520., XP002912486, ISSN: 0021-9258, DOI: 10.1074/jbc.271.1.512 * |
See also references of EP0972013A4 * |
TAKAHASHI H., LIZUKA H.: "ANALYSIS OF THE 5'-UPSTREAM PROMOTER REGION OF HUMAN INVOLUCRIN GENE: ACTIVATION BY 12-OMICRON-TETRADECANOYLPHORBOL-13-ACETATE.", JOURNAL OF INVESTIGATIVE DERMATOLOGY, NATURE PUBLISHING GROUP, US, vol. 100., no. 01., 1 January 1993 (1993-01-01), US, pages 10 - 15., XP002912488, ISSN: 0022-202X, DOI: 10.1111/1523-1747.ep12349867 * |
WELTER J. F., ET AL.: "REGULATION OF HUMAN INVOLUCRIN PROMOTER ACTIVITY BY POU DOMAIN PROTEINS.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 271., no. 25., 21 June 1996 (1996-06-21), US, pages 14727 - 14733., XP002912487, ISSN: 0021-9258, DOI: 10.1074/jbc.271.25.14727 * |
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CA2284735A1 (en) | 1998-10-01 |
EP0972013A4 (en) | 2002-07-10 |
CA2285413A1 (en) | 1998-10-01 |
AU6767798A (en) | 1998-10-20 |
JP2001517956A (en) | 2001-10-09 |
EP0972013A1 (en) | 2000-01-19 |
WO1998042825A1 (en) | 1998-10-01 |
AU6869698A (en) | 1998-10-20 |
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