The present invention relates to improved compositions and methods for assaying the efficacy of novel drugs functioning either as agonists or antagonists at nuclear receptors. In particular, the invention provides transgenic non-human animals comprising a human steroid hormone receptor gene operably linked to a promoter which directs expression of the steroid hormone receptor gene in an epithelial cell of the non-human animals.

This invention pertains to the field of transgenic non-human animals. In particular this invention pertains to transgenic animals that express human steroid hormone receptor genes in desired tissues, such as the epithelium, and to methods of screening potential therapeutics for activity at steroid hormone receptors.
Steroid hormones are secreted by the adrenal cortex, testis, ovary and placenta and include the androgens (such as testosterone), estrogens (such as estradiol and estrone), glucocorticoids (cortisone, corticosterone, and cortisol), mineralocorticoids (primarily aldosterone), and progestogens (primarily progesterone). Steroid hormones regulate proliferation and differentiation in target cells within the reproductive tract, mammary gland, and peripheral tissues such as the bones, heart, blood vessels, and hair follicles (for a review, see Yamashita et al., Localization and functions of steroid hormone receptors, 1998, Histol. Histopathol. 13(1):255-70).
Steroid hormones are highly lipophilic and act through nuclear receptors rather than through receptors on the plasma membrane. Steroid hormone receptors have been shown to support the development of cancer in the breast, prostate, uterus, cervix, and ovaries. In addition, steroid hormone receptors appear to prevent osteoporosis of the bones, to prevent atherosclerosis of the coronary arteries of the heart, and to mediate baldness in men. Therefore, steroid hormone nuclear receptors and other gene products that are involved in steroid hormone metabolism are attractive targets for the development of therapeutics that address the treatment of reproductive cancers and conditions such as osteoporosis, atherosclerosis, and baldness.
Steroid hormone receptors are part of a family of nuclear receptors which contain a hormone-biding region and a DNA-binding region, and thereby act as transcription enhancers. Upon binding to their specific ligands, nuclear receptors interact directly with regions of DNA in order to influence transcription of genes regulating hormonal activity. See, Ribeiro et al., The nuclear hormone receptor gene superfamily, 1995, Annual Rev Med. 46:443-53. There is a growing list of drugs that either bind directly to steroid hormone receptors or modulate steroid hormone metabolism. Drugs that bind directly to the nuclear receptors include tamoxifen, an anti-estrogen used in treatment of breast cancer, and raloxifene, used in prevention of osteoporosis. Drugs that modulate sex steroid metabolism include aromatase inhibitors, used to treat breast cancer and possibly prostate cancer, and finasteride (Propecia(trademark)), used in the treatment of hair loss.
Models of targeted expression of oncogenes and growth factors to the epidermis of transgenic mice have been described. These models have used keratin promoters to target the expression of foreign DNA. The basal cell specific keratin-14 (K14) promoter has been used to express growth factors and oncogenes in transgenic animals as models for the development of specific carcinomas (Vassar et al., 1991, Cell 64:365-380; U.S. Pat. No. 5,698,764). There is currently no transgenic model for the expression of human nuclear receptors in the epithelium.
Despite the existence of several drugs that either bind directly to steroid hormone receptors or modulate steroid hormone metabolism, there is a further need to develop models for assaying and testing the site of action and the efficacy of drugs that act to modulate nuclear receptor activity and metabolism. The present invention addresses these and other needs.
The present invention provides transgenic non-human animals exhibiting a detectable phenotype, typically epidermal hyperplasia, caused by a steroid hormone receptor gene operably linked to a promoter which directs expression of the steroid receptor gene in the epithelium in the non-human animal, preferably a mouse. The steroid receptor gene is preferably a human estrogen receptor gene. The promoter used to drive expression of the steroid receptor gene is preferably a keratin-14 promoter directing expression of the steroid receptor in a basal keratinocyte.
The animal may further comprise a xcex2-galactosidase gene operably linked to a promoter which directs expression of the xcex2-galactosidase gene in an epithelial cell. If the steroid hormone receptor gene is expressed in a basal keratinocyte, the xcex2-galactosidase gene is also preferably expressed in a basal keratinocyte.
The invention also provides DNA constructs comprising an expression cassette including a steroid hormone receptor gene operably linked to a promoter which directs expression of the steroid hormone receptor in the epithelium, preferably in a basal keratinocyte. The promoter is preferably the keratin-14 promoter.
The invention further provides methods of testing a composition for the ability to modulate steroid hormone receptor activity. The methods comprise providing a transgenic non-human animal comprising a human steroid hormone receptor gene operably linked to a keratin-14 promoter which directs expression of the gene in an epithelial cell, administering the composition to the non-human animal, and detecting changes in the epithelial cell of the non-human animal.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.
As used herein, xe2x80x9cnon-human animalsxe2x80x9d include, for example, mammals such as non-human primates, ovine, canine, bovine, rattus and murine species, as well as rabbits and the like. Preferred non-human animals are selected from the rodent family, including rat, guinea pig and mouse, most preferably mouse.
As used herein, a xe2x80x9csteroidxe2x80x9d or xe2x80x9csteroid hormonexe2x80x9d refers to any one of a group of biologically active compounds synthesized from cholesterol that contain a cyclopentanoperhydrophenanthrene nucleus. A xe2x80x9csteroid receptorxe2x80x9d or xe2x80x9csteroid hormone receptorxe2x80x9d is a nuclear receptor that binds a steroid or steroid hormone described herein.
As used herein, the term xe2x80x9csteroid hormone receptor genexe2x80x9d or xe2x80x9csteroid receptor genexe2x80x9d refers to a nucleotide sequence, or any subsequence thereof, that encodes a steroid receptor described herein or that encodes a gene product exhibiting DNA-binding and steroid hormone-binding activity, in vitro or in vivo; and any conservatively modified variants thereof. Also explicitly included within this definition are both wild-type and mutant genes (e.g. mutant steroid hormone receptor genes isolated from cancer cells) that may or may not have altered activity as compared to wild-type genes.
The term xe2x80x9cestrogen receptorxe2x80x9d refers to a known nuclear receptor having a predicted molecular weight of about 66-kd, that is activated by estrogenic steroid hormones such as estradiol. The active form of the protein enhances expression of genes involved in the formation of secondary sexual characteristics in mammalian females. An estrogen receptor can be an allele, polymorphic variant, interspecies homolog, or any subsequence thereof that exhibits estrogenic steroid hormone-binding activity.
As used herein, xe2x80x9cestrogen receptor genexe2x80x9d is a wild-type or mutant nucleotide sequence that encodes an estrogen receptor described herein, and conservatively modified variants thereof. An example of an estrogen receptor gene is described in Greene et al., 1986, Science 231(4742):1150-4. One of ordinary skill in the art will recognize that certain modifications, additions, and deletions may be made to the estrogen receptor gene sequence which will not affect the function or activity of the gene product. Such variants are included within this definition. An example of a mutant estrogen receptor gene is K206A, in which the lysine at position 206 is replaced by alanine.
The term xe2x80x9coperably linkedxe2x80x9d refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. For example, a promoter is operably linked to a coding sequence if it controls transcription of the sequence.
A xe2x80x9cpromoterxe2x80x9d is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
An xe2x80x9cexpression cassettexe2x80x9d is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression cassette can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
A xe2x80x9cdetectable phenotypexe2x80x9d is any symptom or characteristic of an organism which is measurable by any one or a number of different objective criteria, including, but not limited to, visual inspection, microscopic observation, antibody labeling, DNA labeling, or assays for changes in gene expression which are known in the art. Detectable phenotypes may include, but are not limited to, skin thickening, redness, flaking, epidermal hyperplasia, dysplasia, altered nuclear labeling, or altered protein or gene expression.
The term xe2x80x9cmodulate the activityxe2x80x9d means to have an effect on, e.g., to increase or inhibit or otherwise alter, the activity of, e.g., a human nuclear receptor gene.
The term xe2x80x9cadministeringxe2x80x9d the composition means contacting by any conventional method known to one of skill in the art, such as, for example, parenteral, oral, topical, and inhalation routes.
The phrase xe2x80x9cdetecting changesxe2x80x9d refers to using one or a number of different objective criteria, including, but not limited to, visual inspection, microscopic observation, antibody labeling, DNA labeling, or assays for changes in gene expression which are known in the art, to determine the effect of a composition on a non-human animal of the invention.
xe2x80x9cRecombinantxe2x80x9d refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley and Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.
Two nucleic acid sequences or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 70%, more preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection: This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
One of skill in the art will recognize that two polypeptides can also be xe2x80x9csubstantially identicalxe2x80x9d if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1995 Supplement) (Ausubel)).
Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoffand Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat""l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993). Generally, highly stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Lower stringency conditions are generally selected to be about 15-30xc2x0 C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
In the present invention, genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and at least one wash in 0.2xc3x97SSC at a temperature of at least about 50xc2x0 C., usually about 55xc2x0 C. to about 60xc2x0 C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary xe2x80x9cmoderately stringent hybridization conditionsxe2x80x9d include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and a wash in 1xc3x97SSC at 45xc2x0 C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.
I. Introduction
Steroid hormones are associated with the development of breast, prostate, uterine, cervical, and ovarian cancer. In addition, steroid hormones mediate baldness in men, and are important in the prevention of osteoporosis, and atherosclerosis of the coronary arteries of the heart. Steroid hormone activity is mediated by nuclear steroid hormone receptors. Therefore, steroid hormone receptors and other proteins involved in steroid hormone metabolism are attractive targets for the development and testing of therapeutics that address the treatment of reproductive cancers and conditions such as osteoporosis, atherosclerosis, and baldness.
The present invention provides transgenic animals and DNA constructs for targeting steroid hormone receptor genes to the epithelium of transgenic animals which exhibit an easily observable phenotype. Alternatively, the receptor genes can be targeted to the various tissues in which these cancers occur (e.g. breast, prostate, uterine, cervical and ovarian tissues). These animals, e.g., mice, provide a convenient assay to evaluate the effect of compositions that function either as agonists or antagonists of the target steroid hormone receptors and metabolizing enzymes. The present invention is thus useful in the testing of potential therapeutic drugs that target either steroid hormone receptors or other genes that are involved in the metabolism of steroid hormones, e.g, genes encoding enzymes such as aromatase. For example a test compound can first be evaluated in a transgenic animal in which the steroid hormone receptor gene is targeted to the epithelium and then tested further in an animal in which the gene is targeted to a desired tissue, e.g. breast, in the case of screens for compounds useful in treating breast cancer.
Generally, the nomenclature used hereafter and the laboratory procedures in molecular genetics described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgene incorporation (e.g., electroporation, microinjection, lipofection). Generally enzymatic reactions, oligonucleotide synthesis, and purification steps are performed according to the manufacturer""s specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general and specific references which are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. Much of the nomenclature and general laboratory procedures described below can be found in Sambrook, et al., Molecular Cloningxe2x80x94A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
II. DNA Constructs
Appropriate constructs for production of vectors used to make transgenic animals are described in Hogan et al., 1986, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. In the construction of vectors for the production of transgenic animals, the coding sequence of interest is typically operably linked to expression regulatory sequences. In such transgenes, the expression regulatory sequence is at least the minimal sequences required for efficient cell-type specific expression, which generally are at least a promoter and at least about 1 kilobase (kb) upstream of the promoter. Usually the sequences upstream of the promoter are used contiguously, although various deletions and rearrangements can be employed. Some desired regulatory elements (e.g., enhancers, silencers) may be relatively position-insensitive, so that the regulatory element will function correctly even if positioned differently in a transgene than in the corresponding germline gene. For example, an enhancer may be located at a different distance from a promoter, in a different orientation, and/or in a different linear order. For example, an enhancer that is located 3xe2x80x2 to a promoter in germline configuration might be located 5xe2x80x2 to the promoter in a transgene.
Typically, expression regulation sequences are chosen to produce tissue-specific or cell type-specific expression of the desired structural gene. In the present invention the targeted cells are, e.g., breast, prostate, uterine, cervical, ovarian or epithelial cells. Once a tissue or cell type is chosen for expression, expression regulation sequences are chosen. Generally, such expression regulation sequences are derived from genes that are expressed primarily in the tissue or cell type chosen. Preferably, the genes from which these expression regulation sequences are obtained are expressed substantially only in the tissue or cell type chosen, although secondary expression in other tissue and/or cell types is acceptable if expression of the recombinant DNA in the transgene in such tissue or cell type is not detrimental to the transgenic animal.
The constructs will usually also comprise downstream expression regulation sequences to supplement tissue or cell-type specific expression. The downstream expression regulation sequences include polyadenylation sequences (either from the endogenous gene or from other sources such as SV40) and sequences that may affect RNA stability as well as enhancer and/or other sequences which enhance expression.
In some embodiments of the present invention, a steroid hormone receptor gene is placed in an expression cassette under the control of a promoter that will direct expression of the gene to an epithelial cell. A number of suitable promoters can be used to direct expression of the steroid hormone nuclear receptor gene in epithelial cells. Particularly useful for targeting the expression of sequences to epithelial cells are the promoters from genes encoding keratin. Keratins are proteins that are expressed in epithelial tissues. Specific keratin proteins, identified by a number, e.g., keratin-5, are exclusively expressed not only in certain epithelia, but also in selected cells populating the epithelia. The epidermis is composed of layers of cells (keratinocytes) which produce specific types of keratin proteins. The basal cells produce keratin 5 and 14 (K5 and K14), whereas the more mature, terminally differentiated keratinocytes, e.g., the suprabasal keratinocytes, produce K10 and K1. Promoters from other keratin genes, such as K8 and K19 are useful in directing expression to epithelia in the bladder or intestines.
In a preferred embodiment, a basal cell keratin promoter (e.g., K5 or K14) is utilized. The K14 promoter is particularly preferred. A K14 expression cassette, containing 2 kb of the K14 promoter/enhancer and 500 bp of the 3xe2x80x2 flanking sequence including the K14 polyadenylation signal, has been shown to appropriately target expression of transgenes to the basal cells of squameous epithelium (Vassar et al., 1991, Cell 64:365-380; Cheng et al., 1992 Genes Dev. 6:1444-1456; Guo et al., 1993, EMBO J. 12:973-986; Turksen et al., 1992, Proc. Natl. Acad. Sci. USA 89:5068-5072; Vassar et al., 1989, Proc. Natl. Acad. Sci. USA 86:1563-1567; U.S. Pat. No. 5,698,764). This cassette is preferred for construction of the transgene of the present invention.
Alternatively, the steroid hormone receptor genes can be targeted to other tissues, such as breast, prostate, uterine, cervical, and ovarian tissue. Promoters for expression in these tissues are known to those of skill. Examples of suitable promoters include the mouse mammary tumor virus (MMTV) promoter (see, e.g. Guy et al. Mol. Cell. Biol. 12:954-961 (1992)) for expression in breast tissue and the probasin promoter for expression in prostate cells (see, e.g. Yan et al. Prostate 32:129-130 (1997))/
In preferred embodiments, steroid hormone receptor genes are used in the DNA constructs of the invention. One of skill will recognize that the genes need not be naturally occurring wild-type genes, but can be conservatively modified variants or mutants. The human estrogen receptor (ER) gene is conveniently used. See Greene et al., Sequence and expression of human estrogen receptor complementary DNA, 1986, Science 231(4742):1150-4. One mutant ER gene that can be used is the K206A mutant, in which the lysine at position 206 is substituted with alanine. One of skill in the art will recognize that any other steroid hormone receptor gene can be substituted. For example, the androgen, progesterone, mineralocorticoid, and glucocorticoid receptor genes can conveniently be substituted. See, e.g., Lubahn et al., The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate, 1988, Mol Endocrinol. 2(12):1265-75; Misrahi et al., Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA, 1987, Biochem Biophys Res Commun. 143(2):740-8; Arriza et al., Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor, 1987, Science 237(4812):268-75; Govindan et al., Cloning of the human glucocorticoid receptor cDNA, 1985, Nucleic Acids Res. 13(23):8293-304.
Further, genes encoding metabolizing enzymes that regulate steroid hormones are suitable targets for the DNA constructs and transgenic animals of the invention. For example, the gene encoding aromatase, an enzyme that converts androgens to estrogen, is conveniently substituted. See, Harada et al., Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis, 1988, Biochem Biophys Res Commun. 156(2):725-32. Aromatase inhibitors are used to treat breast cancer patients. The recent discovery of a distinct gene for another estrogen receptor (estrogen receptor-beta) suggests that prostate cancer may respond to aromatase inhibitors as well. Chang et al., Estrogen receptor-beta: implications for the prostate gland, 1999, Prostate 40(2):115-24.
Methods of modifying the DNA constructs of the invention are well known to those of skill in the art (see, for example, Sambrook et al., 1989 supra; Methods in Enzymology, 1987, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, eds., San Diego, Academic Press, Inc.; or Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, N.Y.).
III. Production of Transgenic Animals
The transgenic non-human animals of the invention are produced by introducing expression cassettes comprising the desired promoted and structural gene (e.g. K14-estrogen receptor transgene) into the germline of the non-human animal. In a preferred embodiment, the transgenic non-human animal is selected from the rodent family, including rat, guinea pig and mouse. Most preferably, the transgenic animal is a mouse. The transgenic animal may also be a non-human primate, a member of the ovine, canine, or bovine species, or a rabbit and the like.
In preferred embodiments two expression cassettes are introduced into the animal. One cassette comprises the recombinant expression cassette of the invention and the other comprises a selectable marker gene such as xcex2-galactosidase or xcex2-glucuronidase (GUS), useful in monitoring expression of the transgene. Genes encoding xcex2-galactosidase are particularly preferred. Embryonic target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonic target cell.
A. MicroInjection Methods
Microinjection is a preferred method for transforming a zygote or early stage embryo. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pl of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will, in general, also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
The gene sequence being introduced need not be incorporated into a self-replicating plasmid or virus (Jaenisch, 1988, Science, 240: 1468-1474). However, in a preferred embodiment, the gene sequence will be introduced in a as a cassette comprising the gene under the control of a promoter. The promoter acts to regulate transcription of the gene in response to endogenous factors present in a particular tissue and thus results in tissue-specific expression of the gene.
Once the DNA molecule has been injected into the fertilized egg cell, the cell is implanted into the uterus of a recipient female, and allowed to develop into an animal. Since all of the animal""s cells are derived from the implanted fertilized egg, all of the cells of the resulting animal (including the germ line cells) shall contain the introduced gene sequence. If, as occurs in about 30% of events, the first cellular division occurs before the introduced gene sequence has integrated into the cell""s genome, the resulting animal will be a chimeric animal.
By breeding and inbreeding such animals, it has been possible to produce heterozygous and homozygous transgenic animals. Despite any unpredictability in the formation of such transgenic animals, the animals have generally been found to be stable, and to be capable of producing offspring which retain and express the introduced gene sequence.
The success rate for producing transgenic animals is greatest in mice. Approximately 25% of fertilized mouse eggs into which DNA has been injected, and which have been implanted in a female, will become transgenic mice. A number of other transgenic animals have also been produced. These include rabbits, sheep, cattle, and pigs (Jaenisch, 1988, Science 240: 1468-1474; Hammer et al., 1986, J. Animal Sci, 63: 269; Hammer et al., 1985, Nature 315: 680; Wagner et al., 1984 Theriogenology 21: 29).
B. Retroviral Methods
Retroviral infection can also be used to introduce a transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, 1976, Proc. Natl. Acad. Sci USA 73: 1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al., 1986, In Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector system used to introduce the transgene is typically 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 easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra ; Stewart et al., 1987, EMBO J., 6: 383-388). 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). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner et al., 1982, supra).
C. ES Cell Implantation
A third target cell for transgene introduction is the embryonic stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro (Evans et al., 1981, Nature, 292: 154-156; Bradley, et al., 1984, Nature, 309: 255-258; Gossler, et al., 1986, Proc. Natl. Acad. Sci USA 83:, 9065-9069; and Robertson, et al., 1986, Nature, 322: 445-448). Transgenes can be efficiently introduced into ES cells using a number of means well known to those of skill in the art. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (for a review see Jaenisch, 1988, Science, 240: 1468-1474).
In a preferred embodiment, the DNA is introduced by electroporation (Toneguzzo et al., 1988, Nucleic Acids Res., 16: 5515-5532; Quillet et al., 1988, J. Immunol., 141: 17-20; Machy et al., 1988, Proc. Nat""l. Acad. Sci. USA, 85: 8027-8031). After permitting the introduction of the DNA molecule(s), the cells are cultured under conventional conditions, as are known in the art.
In order to facilitate the recovery of those cells which have received the DNA molecule containing the desired gene sequence, it is preferable to introduce the DNA containing the desired gene sequence in combination with a second gene sequence which would contain a detectable marker gene sequence. For the purposes of the present invention, any gene sequence whose presence in a cell permits one to recognize and clonally isolate the cell may be employed as a detectable (selectable) marker gene sequence. The presence of the detectable (selectable) marker sequence in a recipient cell may be recognized by PCR, by detection of radiolabelled nucleotides, or by other assays of detection which do not require the expression of the detectable marker sequence. Typically, the detectable marker gene sequence will be expressed in the recipient cell, and will result in a selectable phenotype. Selectable markers are well known to those of skill in the art. Some examples include the hprt gene (Littlefield, 1964, Science 145: 709-710), the tk (thymidine kinase) gene of herpes simplex virus (Giphart-Gassler et al., 1989, Mutat, Res., 214: 223-232), the nDtII gene (Thomas et al., 1987, Cell, 51: 503-512; Mansour et al., 1988, Nature 336: 348-352), or other genes which confer resistance to amino acid or nucleoside analogues, or antibiotics, etc.
Any ES cell may be used in accordance with the present invention. It is, however, preferred to use primary isolates of ES cells. Such isolates may be obtained directly from embryos such as the CCE cell line disclosed by Robertson, E. J., 1989, In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 39-44), or from the clonal isolation of ES cells from the CCE cell line (Schwartzberg et al., 1989, Science 212: 799-803). Such clonal isolation may be accomplished according to the method of Robertson, 1987, In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, Ed., IRL Press, Oxford. The purpose of such clonal propagation is to obtain ES cells which have a greater efficiency for differentiating into an animal. Clonally selected ES cells are approximately 10-fold more effective in producing transgenic animals than the progenitor cell line CCE.
D. Verification of the Presence of the Transgene in the Animal
A number of methods used to detect particular polynucleotide sequences can be used to verify that the desired sequences have been integrated into the genome of the transgenic animal. For instance, Fluorescent In Situ Hybridization (FISH) can be used to detect the transgene in tissue from the animal. Several guides to FISH techniques are available, e.g., Gall et al,. 1981, Meth. Enzymol., 21:470-480, and Angerer et al., 1985, in Genetic Engineering: Principles and Methods Setlow and Hollaender, Eds. Vol 7, pg. 43-65. Alternatively, DNA or RNA can be isolated for tissue (typically tail tissue). The desired sequences can be detected by Southern or Northern hybridization or by PCR using primers and probes specific for the transgene. Standard PCR methods useful in the present invention are described in PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., Academic Press, San Diego 1990).
Alternatively, expression of the integrated gene can also be detected by detecting the gene product. The protein can be detected, for instance, using standard immunoblotting techniques, well known to those of skill in the art.
IV. Methods to Evaluate the Efficacy of Compositions Functioning at Steroid Hormone Receptors
A valuable feature of the transgenic animals of the invention is an easily observable phenotype in the epidermis of the transgenic animal. In some embodiments, the non-human transgenic animals exhibit epidermal hyperplasia. They may alternatively, or in addition, exhibit other phenotypes, including, but not limited to, thickening (acanthosis), redness (erythema), or flaking (hyperkeratosis). Therefore, the transgenic animals of the invention are useful in methods for screening drugs and treatments designed to target nuclear steroid hormone receptors and other proteins involved in steroid hormone metabolism.
The methods herein provided for testing a composition comprise providing a transgenic non-human animal comprising a human steroid hormone receptor gene operably linked to a keratin-14 promoter, administering the composition to the non-human animal, and detecting changes in the epidermis of the non-human animal. The compositions that are tested by this method will depend on the target transgene of the transgenic animal. For example, the K14-estrogen receptor mouse of the invention can be used to assay the efficacy of anti-estrogen drugs, such as tamoxifen, raloxifen, and the like. Similarly, a transgenic animal expressing a K14-androgen receptor transgene in hair follicles can be used to assay drugs directed at androgen receptors for the treatment of baldness. Still further, a transgenic animal comprising a transgene encoding an enzyme involved in steroid hormone metabolism can be used to assay drugs thought to modulate sex steroid metabolism. Examples of such drugs include aromatase inhibitors, and fenesteride (Propesia(trademark)), which blocks an enzyme that converts testosterone into the related hormone dihydrotestosterone.
In a preferred embodiment, the epithelium of the epidermis of the transgenic animal is used as a readout to detect changes in the transgenic animal and evaluate the efficacy of compositions functioning either as agonists or antagonists at nuclear receptors and other proteins involved in steroid hormone metabolism. The advantage of using the epidermis as an assay is that it can be conveniently evaluated by a number of different visual and microscopic objective criteria, as well as by molecular assays. For example, the epidermis can be visually inspected for changes such as thickening (acanthosis), redness (erythema), and flaking (hyperkeratosis). In addition, the skin can be evaluated microscopically for hyperplastic thickening and alterations of appearance which are consistent either with normal differentiation, or with dysplasia, such as the persistence of immature neoplastic cells in the upper epidermal layers. At the molecular level, panels of keratin antibodies exist which can identify the keratin expression patterns of transgenic versus non-transgenic epidermis, as each layer of the epidermis produces a repertoire of keratin intermediate filaments specific for cell types within that layer. Still further, the DNA labeling pattern of epidermis is distinct and can be used to compare the number of labeled nuclei from transgenic and non-transgenic epidermis.

Expression of human estrogen receptors in transgenic mice

The Regents of the University of California


A buffering protocol and a streaming media player which utilizes the buffering protocol designed for minimizing the occurrences of pauses during the presentation due to network congestion and for scheduling pauses at appropriate times in the presentation. The data comprises coded content information comprising a time synchronous media, together with various other sources, synchronized to appear at predetermined time points in the media presentation. The presentation is delivered in streaming fashion, so that the end user does not have to wait for the entirety of the data to be downloaded before starting the presentation. The invention does not require that media data and events data be multiplexed into a single streaming file and it does not require that the file provided to the media player be customized for prefetching of content.

The invention relates to the provision of multimedia presentations at a user computer and more particularly to a streaming media player capable of synchronizing content from different sources into a multimedia presentation for display at the user location.
The Internet and various intranets are well known communication networks for the transfer of digital data. While most of the data transmitted on these networks correspond to text or certain computer programs, more and more of it now pertains to multimedia content such as images, audio and video.
A hypertext document is one which is linked to other documents via hyperlinks. A hyperlink often appears in a hypertext document as a piece of highlighted text. The text is usually a word or phase describing something for which a user might wish to obtain further information. When the user activates the hyperlink, typically by clicking on it using a mouse, a link command is initiated; which causes a program at the linked address to be executed; which, in turn, causes the user""s view to be updated so as to show the linked document, typically containing more information on the highlighted word or phase. Such information may be in the form of text, audio, video, a two-dimensional image or a three-dimensional image. Hyperlinks make it easy to follow cross-references between documents. Hypermedia documents are hypertext documents with multimedia capabilities. The regions on the screen which are active hyperlinks are called hot-links.
Nowadays, most people are familiar with the application of hypertext by using a mouse to click on a hot-ink provided on a computer display of a homepage from the World Wide Web (the Web) on the Internet. Data on the Web is located via Uniform Resource Locators, or URLs. URLs comprise the draft standard for specifying an object on the Internet. Each URL specifies the access method and the location for the files. Documents on the Web are written in a simple xe2x80x9cmarkup languagexe2x80x9d called HTML, which stands for Hypertext Markup Language. File formats of data on the Web are specified as MIME formats; MIME stands for xe2x80x9cMultipurpose Internet Mail Extensions.xe2x80x9d (Reference: http://www.oac.uci.edu/indiv/ehood/MIME/MIME.html). Examples of file formats on the Web are .au (probably the most common audio format), .html (HTML files), .jpg (JPEG encoded images), .mid (Midi music format), mpg (MPEG encoded video), and .ps (postscript files). While presently hypertext technology is most common in text and image media, it is beginning to also appear in animation, video and audio.
Audio is also encoded in wav format and stored in files labeled with the suffix wav. Wav audio is not compressed beyond the quantization due to sampling rate and bits per sample. Radio quality audio is typically 22,050 Hz sampled at 8 bit per channel stereo, which give an encoding at data rates of 43 KBps. Reasonable quality speech can be obtained at 11,025 Hz sampling, 8 bit mono, yielding data rates of 11 KBps. MPEG provides various standards for audio compression, typically derived from 44,100 Hz sampling stereo at 16 bit per sample. MPEG audio is typically compressed to between 16 Kbps to 384 Kbps. Other standards tailored to speech signals compress to as 5 Kbps; these include G.723 and GSM.
Typical Web servers follow the HTTP protocol. When a user requests the content of a URL on a server, the entire content associated with that URL is sent to the user""s client machine. Such content may be comprised of an html or htm document with auxiliary information attached to it, such as images and perhaps animation software. The server will commence sending the data and continue sending same until either it has completed sending all the data or until it has received a message from the client to stop sending any more data. Some servers serve in streaming mode, wherein data is sent at some prescribed average data rate, say K bits every N seconds. A streaming server is serviced by a scheduling algorithm to maintain this average data rate.
Media players for decoding and playing audio and video have been standard features on personal computers for more than a decade. Apple Computers had their QuickTime player, while machines running Microsoft""s Windows operating system had the Microsoft Media Player. The players typically required that data for the entire content to be played be resident locally on the computer before the player could start playing. This meant that when media content was coming from some other source on the Web, the player would have to wait until all content was downloaded before starting to play. Recently media players have begun to support streaming capabilities. Streaming players buffer some data from outside sources on the Web and then start playing, even though much of the data has not yet arrived. If the data rate of the incoming data is not fast enough, the player would pause when the data in its buffer was depleted, rebuffer with more data, and then continue to play. An improved streaming media player is detailed in co-pending patent application Ser. No: 09/293,644, entitled xe2x80x9cVariable Rate Streaming Media Playerxe2x80x9d, which is assigned to the present assignee, wherein the media player can slow its playback if it detects a decrease in the buffering of incoming media content, based on network delays, etc.
Streaming media have found novel new applications. One such application is the delivery of audio presentations augmented with images or transparencies. The images are displayed at appropriate time intervals during the audio playback, as prescribed by the authors of the presentation. Various technologies have been invented to accommodate such presentations. Real Networks is using a file format called SMIL, which encapsulates all the relevant information in one file, makes certain that all the data that is required to be provided at a particular point in a presentation already be present at the client at such instant, and then streams this file using a streaming server at some prescribed data rate. Microsoft""s NetShow utilizes a similar scheme but with its ASF data format. All known techniques for delivery of such synchronized content utilize a multiplexing of all the content into a single file, followed by streaming that file using a streaming server. Often, however, the two requirements of a single file and a streaming server are undesirable added complexities. An invention on which the present authors have filed a co-pending patent application, Ser. No. 04/396,241, entitled xe2x80x9cEfficient Streaming of Synchronized Web Content From Multiple Sourcesxe2x80x9d filed simultaneously herewith and assigned to the present assignee, provides a solution without the two requirements, but needed special authoring to prepare auxiliary information to be sent to client players which are then utilized to schedule the various requests to the servers.
What is desirable, therefore, and what is an object of the invention, is to provide a system and method to enable the presentation of time synchronous content without the requirements of creating a single file, including a streaming server, or providing auxiliary information regarding the scheduling of requests.
Another object of the invention is to provide the foregoing in a system and method which requires only input of the time points in the presentation at which various images and events need to occur.
Yet another object of the invention is to provide the foregoing even if the various files do not reside on the same server.
These and other objects are realized by the present invention which provides for the delivery of data from one or more sources, typically web servers, over a communications network such as the Web or an intranet, to end users typically deploying computers. The invention describes a buffering protocol and a streaming media player which utilizes the buffering protocol designed for minimizing the occurrences of pauses during the presentation due to network congestion and for scheduling pauses at appropriate times in the presentation. The data comprises coded content information comprising a time synchronous media, such as audio or video, together with various other sources, such as images or events to be displayed on a monitor, synchronized to appear at predetermined time points in the media presentation. For example, the data may comprise all the information required for the presentation of a lecture using audio and images of accompanying transparencies, each transparency displayed at an appropriate interval of time during the audio presentation. The presentation is delivered in streaming fashion, so that the end user does not have to wait for the entirety of the data to be downloaded before starting the presentation, but rather can start viewing and listening to the presentation after a relatively short delay period. In case of network congestion, as if often experienced on the Web, some of the data necessary for the presentation at a particular instant of time might not yet have arrived, which causes the presentation to pause until the necessary data has arrived. Without the buffering protocol, such pauses will be arbitrary and perhaps frequent. The buffering protocol is ideally suited for streaming media players that do not utilize special streaming servers. The invention does not require that media data and events data be multiplexed into a single streaming file and it does not require that the file provided to the media player be customized for prefetching of content.

Streaming media player with synchronous events from multiple sources

International Business Machines Corporation


An inflatable positioning device is provided. The device includes a pump, a tube extending from the pump, a valve intermediate the length of the tube and non-rectangular inflatable pillow connected to the end of the tube remote from the pump. The non-rectangular inflatable pillow is dimensioned for positioning portions of a patient""s body during surgery.

1. Field of the Invention
The subject invention relates to inflatable positioning aids for an operating room that enable a patient to be conveniently and safely positioned during surgery.
2. Description of the Prior Art
An anesthetized patient must be maintained substantially immovably in a position that enables convenient access by doctors during surgery. Additionally, the anesthetized patient must be positioned in a manner that maintains an open air passage for continuous unstressed breathing. Still further, the anesthetized patient must be positioned in a manner that will prevent long term pressure at a location that could impede blood flow or put pressure on nerves or vital structures.
Doctors and other members of a surgical team continue to use fairly crude positioning devices during surgery. For example, patients may be propped into a selected position by rolled-up sections of blankets, sheets and gauze pads. However, these fairly crude positioning devices do not provide an acceptable degree of control, and do not facilitate repositioning intraoperatively. Furthermore, a roll of fabric, or the like, may create local pressure points that would not be appreciated by the surgical team and that could impede blood flow. A restriction of blood flow during a lengthy operation can cause serious damage to limbs, and hence can require a long term regimen of post-operative physical therapy. Nerve damage or damage to other organs may be permanent.
The prior art has included several inflatable positioning aids. Most of the prior art inflatable positioning aids are very complicated structures that would be costly to manufacture. The complexities arise from having a plurality of angularly aligned internal or external panels that must be carefully secured to one another. The prior art requirement for the plurality of angularly aligned, intersecting and seamed panels adds to manufacturing and assembly costs and substantially complicates quality control inspections of the product.
The assignee of the subject invention also has rights to an inflatable positioning aid. The assignee""s inflatable positioning aid is described in copending application Ser. No. 08/733,629 which was filed on Oct. 17, 1996, and includes: a generally rectangular inflatable pillow; a tube extending into the pillow; a hand pump for directing air through the tube and into the pillow; and a valve for selectively releasing air from the pillow.
Despite the many advantages of the assignee""s prior inflatable positioning aid, the need for further improvements has been recognized by the inventors herein. For example, certain types of surgery create unique positioning requirements that may not be met appropriately by a rectangular inflatable pillow.
The subject invention relates to solutions to certain of those unique surgical positioning demands.
The subject invention is directed to inflatable positioning aids for use during a medical procedure. More particularly, the subject invention relates to inflatable positioning aids that are uniquely configured to meet demands of certain medical procedures. These inflatable positioning aids may be used with a flexible tube, a hand pump for manually inflating the positioning aid and a valve for selectively deflating the positioning aid. The inflatable positioning aid also may be used with the tube, the valve and an adaptor that can be engaged with the flexible tube and with a source of compressed air for automatic inflation of the positioning aid. Such a source of compressed air commonly is present in an operating room. Thus, the use of a source of a compressed air enables very rapid inflation and avoids the need to have a person in the operating room mechanically pump air into the inflatable positioning aid.
In all embodiments, the inflatable positioning aid is formed from a top panel and a bottom panel, at least portions of which are of substantially identical shape. Preferred embodiments of the inflatable positioning aids of the subject invention do not include end panels, side panels or internal panels that would complicate assembly and increase costs. Rather, the desired shape of the inflatable positioning aid is achieved by the careful selection of the non-inflated shapes of the top and bottom panels as described herein. In all embodiments, the inflatable positioning aid is assembled by positioning identically configured portions of the top and bottom panels in registration with one another and seaming the top and bottom panels to one another at selected locations.
At least one of the panels of the positioning aid may include a non-inflatable flap extending beyond the periphery of the inflatable portions of the positioning aid. The flap is disposed at a location on which a portion of the patient will lie. Thus, the flap functions to hold the inflatable positioning aid at a specified location during inflation, and during a medical procedure.
In one embodiment, the inflatable positioning aid defines a wedge-shape after inflation that enables the patient to be shifted from a perfectly horizontal supine position into a position where one side of a patient is elevated slightly. The inflated positioning aid to meet these requirements may be tapered from a first end defining a point or line of substantially zero cross-section to a second end defining a circle, oval or rectangle of larger cross-section. The cross-sectional difference between the first and second ends can be varied by the sizes and shapes of the panels and by the amount of air pumped into the inflatable portion of the positioning aid. Positioning aids of this type will have particularly utility in certain obstetric procedures, such as a caesarian section. In particular, during all childbirth, the uterus tends to compress the vena cava and/or the aorta, thereby impairing blood flow to the fetus (aorto-caval compression syndrome). The shifting enabled by the wedge-shape after inflation displaces the uterus and helps to avoid the compression of the vena cava and/or aorta. The inflatable positioning aid may have a non-inflatable flap extending from one end. The flap may be placed under the patient to prevent lateral shifting as the wedge-shape develops during inflation and to prevent shifting during childbirth.
The subject invention also relates to a non-rectangular inflatable positioning aid intended for supporting the face in surgical procedures that require a patient to be in a prone position. In this position, it is often difficult to conveniently position the face in a manner that will ensure that the air passages remain open. Accordingly, a generally U-shaped inflatable positioning aid may be provided. The U-shaped inflatable portion of the positioning aid may have an inflatable connecting portion and a pair of inflatable arms. The opening between the pair of inflatable arms may be dimensioned to receive the nose and mouth of the patient. In certain embodiments, the opening between the arms may be substantially T-shaped, with the central portion of the T being dimensioned and configured for receiving the nose and mouth, and with the arms of the T being dimensioned and configured for receiving the eyes of the patient, to prevent excessive pressure on the eyes. The inflatable positioning aid of this embodiment may be made of a transparent material to enable the anesthesiologist to clearly see the face and eyes when the patient is in the prone position. Ends of the arms of the inflatable positioning aid remote from the connecting portion may be joined by a sheet member or strap for ensuring that the inflatable arms remain in selected positions relative to one another, and to prevent the inflatable arms from spreading in a manner that would urge the nose and mouth downwardly against the operating table. A non-inflatable flap may further extend from ends of the arms of the inflatable positioning aid remote the connecting portion. The non-inflatable flap will carry the weight of the torso of the patient and will ensure that the inflatable positioning aid does not shift significantly relative to the patient in response to forces generated during inflation or during the medical procedure. A similar configuration may be employed for back surgery. In this embodiment, the inflatable arms diverge in a V-shape. Additionally, the non-inflatable flap may extend from the inflatable connecting portion. The space between the inflatable arms may be positioned to align with the sternum, and the non-inflatable flap may be positioned under the hips.
A third inflatable positioning aid is elongated and dimensioned to extend over substantially the entire torso of a patient. This third inflatable positioning aid includes a generally rectangular top panel and a rectangular bottom panel. The rectangular top and bottom panels may be secured to one another about the periphery of at least one panel. Additionally, an elongated central portion is rendered non-inflatable by securing the central portion of the top panel to the central portion of the bottom panel. Areas of the central portion that are not inflated may extend continuously from one longitudinal end of the rectangular inflatable positioning aid to a location near the opposed longitudinal end. This will create first and second spaced apart longitudinally extending inflatable portions. Additionally, the first and second longitudinally extending inflatable portions are connected to one another at a location in proximity to at least one of the ends. Thus, a single source of air can be employed to inflate both longitudinally extending sections of the inflatable position aid. The non-inflated central portion of this inflatable positioning aid preferably is disposed to substantially align with the spine of the patient. Thus, the spine will extend along the non-inflated groove and will not be subjected to pressure. Furthermore, this positioning aid provides two elongate inflatable supports that ensure uniform stable elevation without rocking or tilting that could occur with a single rectangular chamber under the back. An inflatable positioning aid of this type is particularly useful for heart surgery. To ensure that the inflatable positioning aid does not move, this embodiment may include first and second non-inflatable flaps extending respectively from the opposed longitudinal ends of the inflatable portion. The first non-inflatable flap may be positioned under the hips of the patient, and the second non-inflatable flap may be positioned under the head of the patient. Each flap may be a unitary extension of one panel.
A fourth inflatable positioning aid in accordance with the subject invention is intended for positioning the neck and head when the patient is in a supine position. More particularly, this inflatable positioning aid may include a generally toroidal section dimensioned and configured for supporting the back of the head and an elongate portion for positioning under the neck. The elongate portion may be substantially tangential to the toroidal portion. Additionally, the toroidal portion and the elongate portions may be separately controllable. Thus, the valve employed in this embodiment may differ from the valve employed in other embodiments in that air pressure may be selectively added to or withdrawn from either of the two separate sections of the inflatable support in accordance with the particular needs of the patient.

Inflatable positioning aids for operating room