WO2002066614A2 - Chimeric retinoid x receptors and their use in a novel ecdysone receptor-based inducible gene expression system - Google Patents
Chimeric retinoid x receptors and their use in a novel ecdysone receptor-based inducible gene expression system Download PDFInfo
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Definitions
- This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates * to a novel ecdysone receptor/chimeric retinoid X receptor-based inducible gene expression system and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.
- Gene expression is a complex biological process involving a number of specific protein-protein interactions.
- a transcriptional activator In order for gene expression to be triggered, such that it produces the RNA necessary as the first step in protein synthesis, a transcriptional activator must be brought into proximity of a promoter that controls gene transcription.
- the transcriptional activator itself is associated with a protein that has at least one DNA binding domain that binds to DNA binding sites present in the promoter regions of genes.
- a protein comprising a DNA binding domain and a transactivation domain located at an appropriate distance from the DNA binding domain must be brought into the correct position in the promoter region of the gene.
- the traditional transgenic approach utilizes a cell-type specific promoter to drive the expression of a designed transgene.
- a DNA construct containing the transgene is first incorporated into a host genome. When triggered by a transcriptional activator, expression of the transgene occurs in a given cell type.
- inducible promoters Another means to regulate expression of foreign genes in cells is through inducible promoters.
- inducible promoters include the PRl-a promoter, prokaryotic repressor- operator systems, immunosuppressive-immunophilin systems, and higher eukaryotic transcription activation systems such as steroid hormone receptor systems and are described below.
- the PRl-a promoter from tobacco is induced during the systemic acquired resistance response following pathogen attack.
- the use of PRl-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants.
- Gene regulation systems based on promoters induced by heat shock, interferon and heavy metals have been described (Wurn et al., 1986, Proc. Natl.
- Prokaryotic repressor-operator systems utilize bacterial repressor proteins and the unique operator DNA sequences to which they bind. Both the tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systems from the bacterium Escherichia coli have been used in plants and animals to control gene expression. In the Tet system, tetracycline binds to the TetR repressor protein, resulting in a conformational change which releases the repressor protein from the operator which as a result allows transcription to occur. In the Lac system, a lac operon is activated in response to the presence of lactose, or synthetic analogs such as isopropyl-b-D-thiogalactoside.
- Immunosuppressive molecules such as FK506, rapamycin and cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc.
- FK506 rapamycin and cyclosporine A
- FKBP12 immunophilins
- cyclophilin etc.
- a general strategy has been devised to bring together any two proteins simply by placing FK506 on each of the two proteins or by placing FK506 on one and cyclosporine A on another one.
- a synthetic homodimer of FK506 (FK1012) or a compound resulted from fusion of FK506-cyclosporine (FKCsA) can then be used to induce dimerization of these molecules (Spencer et al., 1993, Science 262: 1019-24; Belshaw et al., 1996, Proc Natl Acad Sci U S A 93 :4604-7).
- Gal4 DNA binding domain fused to FKBP 12 and VP 16 activator domain fused to cyclophilin, and FKCsA compound were used to show heterodimerization and activation of a reporter gene under the control of a promoter containing Gal4 binding sites.
- EcR is a member of the nuclear steroid receptor super family that is characterized by signature DNA and ligand binding domains, and an activation domain (Koelle et al. 1991, Cell, 67:59-77).
- EcR receptors are responsive to a number of steroidal compounds such as ponasterone A and muristerone A. Recently, non-steroidal compounds with ecdysteroid agonist activity have been described, including the commercially available insecticides tebufenozide and methoxyfenozide that are marketed world wide by Rohm and Haas Company (see International Patent Application No. PCT/EP96/00686 and US Patent 5,530,028). Both analogs have exceptional safety profiles to other organisms.
- retinoid X receptor retinoid X receptor
- EcR insect ecdysone receptor heterodimerizes with retinoid X receptor (RXR) and regulates expression of target genes in a ligand dependent manner.
- PCT/US98/14215 discloses that the ecdysone receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner.
- U.S. Patent No. 5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins.
- USP Drosophila melanogaster EcR and ultraspiracle
- a chimeric RXR ligand binding domain comprising at least two polypeptide fragments, wherein the first polypeptide fragment is from one species of vertebrate/invertebrate RXR and the second polypeptide fragment is from a different species of vertebrate/invertebrate RXR, whereby a vertebrate/invertebrate chimeric RXR ligand binding domain, a vertebrate/vertebrate chimeric RXR ligand binding domain, or an invertebrate/invertebrate chimeric RXR ligand binding domain is produced, can function similar to or better than either the parental vertebrate RXR or the parental invertebrate RXR in an ecdysone receptor-based inducible gene expression system.
- Applicants' novel ecdysone receptor/chimeric retinoid X receptor-based inducible gene expression system provides an inducible gene expression system in bacteria, fungi, yeast, animal, and mammalian cells that is characterized by increased ligand sensitivity and magnitude of transactivation.
- the present invention relates to a novel ecdysone receptor/chimeric retinoid X receptor-based inducible gene expression system, novel chimeric receptor polynucleotides and polypeptides for use in the novel inducible gene expression system, and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.
- Applicants' invention relates to a novel gene expression modulation system comprising a polynucleotide encoding a chimeric RXR ligand binding domain (LBD).
- the present invention relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an ecdysone receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and ii) a chimeric retinoid X receptor ligand binding domain.
- the present invention also relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first hybrid polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) a chimeric retinoid X receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and ii) an ecdysone receptor ligand binding domain.
- the present invention also relates to a gene expression modulation system according to the invention further comprising c) a third gene expression cassette comprising: i) a response element to which the DNA-binding domain of the first hybrid polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated.
- the present invention also relates to a gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and a chimeric retinoid X receptor ligand binding domain.
- the present invention also relates to an isolated polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and a chimeric vertebrate and invertebrate retinoid X receptor ligand binding domain.
- the present invention also relates to a isolated hybrid polypeptide encoded by the isolated polynucleotide according to the invention.
- the present invention also relates to an isolated polynucleotide encoding a truncated chimeric RXR LBD.
- the isolated polynucleotide encodes a truncated chimeric RXR LBD, wherein the truncation mutation affects ligand binding activity or ligand sensitivity of the chimeric RXR LBD.
- the isolated polynucleotide encodes a truncated chimeric
- RXR polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated chimeric RXR polypeptide and a dimerization partner, hi a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.
- the present invention also relates to an isolated polypeptide encoded by a polynucleotide according to Applicants' invention.
- the present invention also relates to an isolated hybrid polypeptide comprising either i) a DNA- binding domain that recognizes a response element associated with a gene whose expression is to be modulated, or ii) a transactivation domain; and a chimeric retinoid X receptor ligand binding domain.
- the present invention relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation, wherein the truncated chimeric RXR LBD is encoded by a polynucleotide according to the invention.
- the present invention also relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that affects ligand binding activity or ligand sensitivity of said truncated chimeric RXR LBD.
- the present invention also relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated chimeric RXR LBD and a dimerization partner.
- the dimerization partner is an ecdysone receptor polypeptide.
- Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention.
- Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; b) introducing into the host cell a gene expression cassette comprising i) a response element comprising a domain recognized by the DNA binding domain from the first hybrid polypeptide; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated; and c) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host, expression of the gene of b)iii) is modulated.
- Applicants' invention also provides a method of modulating the expression of a gene in a host cell comprising a gene expression cassette comprising a response element comprising a domain recognized by the DNA binding domain from the first hybrid polypeptide; a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and a gene whose expression is to be modulated; wherein the method comprises the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand; whereby upon introduction of the ligand into the host, expression of the gene is modulated.
- Applicants' invention also provides an isolated host cell comprising an inducible gene expression system according to the invention.
- the present invention also relates to an isolated host cell comprising a gene expression cassette, a polynucleotide, or a polypeptide according to the invention.
- Applicants' invention also relates to a non-human organism comprising a host cell according to the invention.
- Figure 1 Expression data of VP16LmUSP-EF, W16MmRXR ⁇ -EF and three independent clones of VP16MmRXR ⁇ (l-7)-LmUSP (8-12)-EF in TH3T3 cells along with GAL4CfEcR-CDEF andpFRLuc in the presence of non-steroid (GSE) ligand.
- GSE non-steroid
- Figure 2 Expression data of VP16LmUSP-EF, W16MmRXR ⁇ -EF and two independent clones of VP16MmRXR ⁇ (l-7)-LmUSP (8-12)-EF inNIH3T3 cells along with GAL4CfEcR-CDEF andpFRLuc in the presence of non-steroid (GSE) ligand.
- Figure 3 Expression data of VP16LmUSP-EF, W16MmRXR ⁇ -EF and two independent clones of VP16MmRXR ⁇ (l-7)-LmUSP (8-12)-EF in A549 cells along with GAL4CfEcR-CDEF and pFRLuc in the presence of non-steroid (GSE) ligand.
- Figure 4 Amino acid sequence alignments of the EF domains of six vertebrate RXRs (A) and six invertebrate RXRs (B).
- B6, B8, B9, BIO and Bl 1 denotes ⁇ chimera junctions.
- Al denotes junction for ⁇ chimera.
- Helices 1-12 are denoted as H1-H12 and ⁇ pleated sheets are denoted as SI and S2.
- F denotes the F domain junction.
- FIG. 5 Expression data of GAL4CfEcR-CDEF/VP16chimeric RXR-based gene switches 1.3-1.6 in NIH3T3 cells along with pFRLuc in the presence of non-steroid (GSE) ligand.
- Figure 6 Expression data of gene switches comprising the DEF domains of EcRs from CfEcR, DmEcR, TmEcR, or AmaEcR fused to GAL4 DNA binding domain and the EF domains of RXR USPs from CfUSP, DmUSP, LmUSP, MmRXR ⁇ , a chimera between MmRXR ⁇ and LmUSP (Chimera), AmaRXRl, or AmaRXR2 fused to a VP16 activation domain along with pFRLuc in NIH3T3 cells in the presence of steroid (PonA) or non-steroid (GSE) ligand.
- PonA steroid
- GSE non-ste
- FIG. 8 Expression data of gene switches comprising the DEF domains of EcRs from CfEcR, DmEcR, TmEcR, or AmaEcR fused to GAL4 DNA binding domain and the EF domains of RXR USPs from CfUSP, DmUSP, LmUSP, MmRXR ⁇ , a chimera between MmRXR ⁇ and LmUSP (Chimera), AmaRXRl, or AmaRXR2 fused to a VP16 activation domain along with pFRLuc in NIH3T3 cells in the presence of steroid (PonA) or non-steroid (GSE) ligand.
- the different EcR constructs were compared in partnership with a chimeric RXR-EF (MmRXR ⁇ -(l-7)-LmUSP(8-12)-EF).
- FIG. 9 Expression data of VP16/MmRXR ⁇ -EF (aRXR), VP16/Chimera between MmRXR ⁇ -EF and LmUSP-EF (MmRXR ⁇ -(l-7)-LmUSP(8-12)-EF; aCh7), VP16/LmUSP-EF (LmUSP) and three independent clones from each of five VP16/chimeras between HsRXR ⁇ -EF and LmUSP-EF (see Table 1 for chimeric RXR constructs; bRXRCh6, bRXRCh.8, bRXRCh9, bRXRChlO, and bRXRChl 1) were transfected into NIH3T3 cells along with GAL4/CfEcR-DEF and pFRLuc. The transfected cells were grown in the presence of 0, 0.2, 1 and 10 ⁇ M non-steroidal ligand (GSE). The reporter activity was quantified 48 hours after adding
- FIG. 10 Expression data of VP16/MmRXR ⁇ -EF (aRXR), VP16/Chimera between MmRXR ⁇ -EF and LmUSP-EF (MmRXR ⁇ -(l-7)-LmUSP(8-12)-EF; aCh7), VP16/LmUSP-EF (LmUSP) and three independent clones from each of five VP16/chimeras between HsRXR ⁇ -EF and LmUSP-EF (see Table 1 for chimeric RXR constructs; bRXRCh ⁇ , bRXRCh ⁇ , bRXRCh9, bRXRChlO, and bRXRChl 1) were transfected into NIH3T3 cells along with GAL4/CfEcR-DEF and pFRLuc.
- the transfected cells were grown in the presence of 0, 0.2, 1 and 10 ⁇ M steroid ligand (PonA) or 0, 0.04, 0.2, 1, and 10 ⁇ M non- steroidal ligand (GSE).
- the reporter activity was quantified 48 hours after adding ligands.
- FIG. 11 Expression data of VP16/MmRXR ⁇ -EF (aRXR), VP16/Chimera between MmRXR ⁇ -EF and LmUSP-EF (MmRXR ⁇ -(l-7)-LmUSP(8-12)-EF; aCh7), VP16/LmUSP-EF (LmUSP) and three independent clones from each of five VP16/chimeras between HsRXR ⁇ -EF and LmUSP-EF (see Table 1 for chimeric RXR constructs; bRXRCb.6, bRXRCh ⁇ , bRXRCh9, bRXRChlO, and bRXRChl 1) were transfected into NIH3T3 cells along with GAL4/DmEcR-DEF and pFRLuc.
- the transfected cells were grown in the presence of 0, 0.2, 1 and 10 ⁇ M steroid ligand (PonA) or 0, 0.04, 0.2, 1, and 10 ⁇ M non- steroidal ligand (GSE).
- the reporter activity was quantified 48 hours after adding ligands.
- Figure 12 Effect of 9-cis-retinoic acid on transactivation potential of the GAL4CfEcR- DEF VP16HsRXR ⁇ -(l-8)-LmUSP-(9-12)-EF ( ⁇ chimera 9) gene switch along with pFRLuc inNIH 3T3 cells in the presence of non-steroid (GSE) and 9-cis-retinoic acid (9Cis) for 48 hours.
- Applicants' invention provides a novel ecdysone receptor-based inducible gene expression system comprising a chimeric retinoid X receptor ligand binding domain that is useful for modulating expression of a gene of interest in a host cell.
- Applicants' invention provides an inducible gene expression system that has a reduced level of background gene expression and responds to submicromolar concentrations of non-steroidal ligand.
- the present invention is useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics, proteomics, and metabolomics analyses and regulation of traits in transgenic organisms, where control of gene expression levels is desirable.
- An advantage of Applicants' invention is that it provides a means to regulate gene expression and to tailor expression levels to suit the user's requirements.
- the term “about” or “approximately” means within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value or range.
- substantially free means that a composition comprising "A” (where "A” is a single protein, DNA molecule, vector, recombinant host cell, etc.) is substantially free of "B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A".
- "A” comprises at least about 90% by weight of the A + B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weiglit species having the activity or characteristic of the species of interest.
- isolated designates a biological material (nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present).
- a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered “isolated”.
- purified does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. It is rather a relative definition.
- a polynucleotide is in the "purified" state after purification of the starting material or of the natural material by at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.
- nucleic acid is a polymeric compound comprised of covalently linked subunits called nucleotides.
- Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both ofwhich may be single-stranded or double-stranded.
- DNA includes but is not limited to cDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA. DNA may be linear, circular, or supercoiled.
- nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
- nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
- this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
- sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
- a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
- fragment will be understood to mean a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid.
- a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide ofwhich it is a constituent.
- Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention.
- an "isolated nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
- An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
- a “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
- “Native gene” refers to a gene as found in nature with its own regulatory sequences.
- “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources.
- “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign" gene or
- heterologous gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
- heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell.
- genomic includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA.
- a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989 infra). Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference).
- Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.
- moderately similar fragments such as homologous sequences from distantly related organisms
- highly similar fragments such as genes that duplicate functional enzymes from closely related organisms.
- low stringency hybridization conditions corresponding to a T m of 55°
- Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
- High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
- Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
- the term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another.
- adenosine is complementary to thymine and cytosine is complementary to guanine.
- the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as disclosed or used herein as well as those substantially similar nucleic acid sequences.
- standard hybridization conditions refers to a T m of 55°C, and utilizes conditions as set forth above, i a preferred embodiment, the T m is 60°C; in a more preferred embodiment, the T m is 65°C.
- Post-hybridization washes also determine stringency conditions.
- One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeated with 2X SSC, 0.5% SDS at 45°C for 30 minutes, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 minutes.
- a more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C.
- Another preferred set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1 % SDS at 65°C.
- Hybridization requires that the two nucleic acids comprise complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
- the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
- the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
- equations for calculating T m have been derived (see Sambrook et al, supra, 9.50-0.51).
- oligonucleotides For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).
- the length for a hybridizable nucleic acid is at least about 10 nucleotides.
- a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides.
- temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
- probe refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.
- oligonucleotide refers to a nucleic acid, generally of at least 18 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
- a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.
- Oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid, or to detect the presence of a nucleic acid.
- oligonucleotide can also be used to form a triple helix with a DNA molecule.
- oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
- a "primer” is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction.
- PCR Polymerase chain reaction
- PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase.
- PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
- RT-PCR Reverse transcription-polymerase chain reaction
- RT-PCR means an in vitro method for enzymatically producing a target cDNA molecule or molecules from an RNA molecule or molecules, followed by enzymatic amplification of a specific nucleic acid sequence or sequences within the target cDNA molecule or molecules as described above.
- RT-PCR also provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
- a DNA “coding sequence” is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
- Suitable regulatory sequences refer to nucleotide sequences located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
- a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3 ' to the coding sequence.
- ORF Open reading frame
- nucleic acid sequence either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.
- head-to-head is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-head orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 5' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds away from the 5' end of the other polynucleotide.
- the term "head-to-head” may be abbreviated (5')-to-(5') and may also be indicated by the symbols ( ⁇ ⁇ ) or (3' ⁇ - 5'5'— >3').
- tail-to-tail is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a tail-to-tail orientation when the 3' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds toward the other polynucleotide.
- the term “tail-to-tail” may be abbreviated (3')-to-(3') and may also be indicated by the symbols (-» -) or (5' ⁇ 3'3' ⁇ -5').
- head-to-tail is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-tail orientation when the 5' end of the coding strand of one polynucleotide is adj cent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds in the same direction as that of the other polynucleotide.
- the term "head-to-tail” may be abbreviated (5')-to- (3') and may also be indicated by the symbols (-> -») or (5'-»3'5'->3').
- downstream refers to a nucleotide sequence that is located 3' to reference nucleotide sequence.
- downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
- upstream refers to a nucleotide sequence that is located 5' to reference nucleotide sequence, hi particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.
- restriction endonuclease and “restriction enzyme” refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.
- homologous recombination refers to the insertion of a foreign DNA sequence into another
- DNA molecule e.g., insertion of a vector in a chromosome.
- the vector targets a specific chromosomal site for homologous recombination.
- the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
- the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.
- a “vector” is any means for the cloning of and/or transfer of a nucleic acid into a host cell.
- a vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment.
- a “replicon” is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
- the term “vector” includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
- vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.
- Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives, or the Bluescript vector.
- the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini.
- the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini.
- Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.
- Viral vectors and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects.
- Viral vectors that can be used include but are not limited to retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors.
- Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.
- a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
- Plasmid refers to an extra chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
- Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been j oined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
- a “cloning vector” is a “replicon”, which is a unit length of a nucleic acid, preferably DNA, that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment.
- Cloning vectors may be capable of replication in one cell type and expression in another ("shuttle vector").
- Vectors may be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g. ,
- a polynucleotide according to the invention can also be introduced in vivo by lipofection.
- liposomes for encapsulation and transfection of nucleic acids in vitro.
- Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 7413; Mackey, et al., 1988, Proc. Natl. Acad. Sci.
- directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain.
- Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al., 1988, supra).
- Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non- peptide molecules could be coupled to liposomes chemically.
- a nucleic acid in vivo such as a cationic oligopeptide (e.g., W095/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., W095/21931). It is also possible to introduce a vector in vivo as a naked DNA plasmid (see U.S. Patents
- Receptor-mediated DNA delivery approaches can also be used (Curiel et al., 1992, Hum. Gene Ther. 3: 147-154; and Wu and Wu, 1987, J. Biol. Chem 262: 4429- 4432).
- transfection means the uptake of exogenous or heterologous RNA or DNA by a cell.
- a cell has been "transfected” by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell.
- a cell has been "transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change.
- the fraiisfom-ing RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
- Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
- genetic region will refer to a region of a nucleic acid molecule or a nucleotide sequence that comprises a gene encoding a polypeptide.
- the recombinant vector comprising a polynucleotide according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.
- selectable marker means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.
- selectable marker genes include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.
- reporter gene means a nucleic acid encoding an identifying factor that is able to be identified based upon the reporter gene's effect, wherein the effect is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and or to measure gene expression induction or transcription.
- reporter genes examples include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase (LacZ), ⁇ -glucuronidase (Gus), and the like. Selectable marker genes may also be considered reporter genes.
- Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
- a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
- Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters”. Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters”. Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
- a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ' direction) coding sequence.
- the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
- a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.
- Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
- polyadenylation signals are control sequences.
- response element means one or more cis-acting DNA elements which confer responsiveness on a promoter mediated through interaction with the DNA-binding domains of the first chimeric gene.
- This DNA element may be either palindromic (perfect or imperfect) in its sequence or composed of sequence motifs or half sites separated by a variable number of nucleotides.
- the half sites can be similar or identical and arranged as either direct or inverted repeats or as a single half site or multimers of adjacent half sites in tandem.
- the response element may comprise a minimal promoter isolated from different organisms depending upon the nature of the cell or organism into which the response element will be incorporated.
- the DNA binding domain of the first hybrid protein binds, in the presence or absence of a ligand, to the DNA sequence of a response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element.
- DNA sequences for response elements of the natural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (see Cherbas L., et. al., (1991), Genes Dev. 5, 120-131); AGGTCAN (n) AGGTCA,where N (n) can be one or more spacer nucleotides (see DAvino PP., et. al., (1995), Mol. Cell. Endocrinol, 113, 1-9); and GGGTTGAATGAATTT (see Antoniewski C, et. al., (1994), Mol. Cell Biol. 14, 4465-4474).
- operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
- a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
- Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
- expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.
- cassette refers to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination.
- the segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
- Transformation cassette refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell.
- Cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell.
- These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.
- the term "gene switch” refers to the combination of a response element associated with a promoter, and an EcR based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated.
- modulate and “modulates” mean to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.
- the plasmids or vectors according to the invention may further comprise at least one promoter suitable for driving expression of a gene in a host cell.
- expression vector means a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host.
- the cloned gene, i.e., the inserted nucleic acid sequence is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like.
- Initiation control regions or promoters which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art.
- Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to: viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoter, developmental specific promoters, inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOXl promoter (useful for expression in Pichia); ⁇ -lactamase, lac, ara, tet, trp, IP jj IPR, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-promoters; animal and mammalian promoters known in the art include, but are not
- Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.
- CMV cytomegalovirus
- EF1 elongation factor 1
- Termination control regions i.e., terminator or polyadenylation sequences, may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.
- the termination control region may be comprise or be derived from a synthetic sequence, synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.
- BGH bovine growth hormone
- 3' non-coding sequences or “3' untranslated region (UTR)” refer to DNA sequences located downstream (3') of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
- the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
- Regulatory region means a nucleic acid sequence which regulates the expression of a second nucleic acid sequence.
- a regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for expressing different proteins or even synthetic proteins (a heterologous region), hi particular, the sequences can be sequences of prokaryotic, eukaryotic, or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific maimer and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcriptional termination sequences, and signal sequences which direct the polypeptide into the secretory pathways of the target cell.
- a regulatory region from a "heterologous source” is a regulatory region that is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.
- RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA.
- Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
- cDNA refers to a double-stranded DNA that is complementary to and derived from mRNA.
- Sense RNA transcript that includes the mRNA and so can be translated into protein by the cell.
- Antisense RNA refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene.
- the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence.
- “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.
- a "polypeptide” is a polymeric compound comprised of covalently linked amino acid residues.
- Amino acids have the following general structure:
- Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
- a polypeptide of the invention preferably comprises at least about 14 amino acids.
- a “protein” is a polypeptide that performs a structural or functional role in a living cell.
- An “isolated polypeptide” or “isolated protein” is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
- “Fragment” of a polypeptide according to the invention will be understood to mean a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence. Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 amino acids.
- a "variant" of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein.
- Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.
- variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin.
- the techniques for obtaining these variants including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
- a variant polypeptide preferably comprises at least about 14 amino acids.
- heterologous protein refers to a protein not naturally produced in the cell.
- a “mature protein” refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
- Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
- signal peptide refers to an amino terminal polypeptide preceding the secreted mature protein.
- the signal peptide is cleaved from and is therefore not present in the mature protein.
- Signal peptides have the function of directing and translocating secreted proteins across cell membranes.
- Signal peptide is also referred to as signal protein.
- a "signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.
- translocation signal sequence is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.
- homology refers to the percent of identity between two polynucleotide or two polypeptide moieties.
- the correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s) and size determination of the digested fragments.
- homologous in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667.). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity. However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as "highly,” may refer to sequence similarity and not a common evolutionary origin.
- sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., 1987, Cell 50:667).
- two DNA sequences are "substantially homologous" or “substantially similar” when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences.
- Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al, 1989, supra.
- substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript.
- substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1 % SDS, 65°C and washed with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS), with the sequences exemplified herein.
- Substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 70% identical to the DNA sequence of the nucleic acid fragments reported herein.
- Preferred substantially nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are at least 90%) identical to the DNA sequence of the nucleic acid fragments reported herein. Even more preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.
- Two amino acid sequences are “substantially homologous” or “substantially similar” when greater than about 40% of the amino acids are identical, or greater than 60% are similar (functionally identical).
- the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program.
- corresponding to is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
- a nucleic acid or amino acid sequence alignment may include spaces.
- corresponding to refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
- a "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215: 403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
- BLAST Basic Local Alignment Search Tool
- a sequence often or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
- gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
- short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
- a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.
- identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
- identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
- Identity and similarity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and
- sequence analysis software refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences.
- Sequence analysis software may be commercially available or independently developed. Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA).
- sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified.
- default values will mean any set of values or parameters which originally load with the software when first initialized.
- Synthetic genes can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene.
- “Chemically synthesized”, as related to a sequence of DNA means that the component nucleotides were assembled in vitro.
- DNA Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be perfo ⁇ ned using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
- the two-hybrid system exploits the ability of a pair of interacting proteins to bring the transcription activation domain into a more favorable position relative to the DNA binding domain such that when the DNA binding domain binds to the DNA binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Patent No. 5,283,173).
- the two-hybrid gene expression system comprises two gene expression cassettes; the first encoding a DNA binding domain fused to a nuclear receptor polypeptide, and the second encoding a transactivation domain fused to a different nuclear receptor polypeptide. the presence of ligand, the interaction of the first polypeptide with the second polypeptide effectively tethers the DNA binding domain to the transactivation domain. Since the DNA binding and transactivation domains reside on two different molecules, the background activity in the absence of ligand is greatly reduced.
- the two-hybrid ecdysone receptor-based gene expression modulation system may be either heterodimeric and homodimeric.
- a functional EcR complex generally refers to a heterodimeric protein complex consisting of two members of the steroid receptor family, an ecdysone receptor protein obtained 5 from various insects, and an ultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid X receptor protein (see Yao, et al. (1993) Nature 366, 476-479; Yao, et al., (1992) Cell 71, 63-72). However, the complex may also be a homodimer as detailed below.
- the functional ecdysteroid receptor complex may also include additional protein(s) such as immunophilins. Additional members of the steroid receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may
- coactivators 10 also be ligand dependent or independent partners for EcR, USP, and/or RXR. Additionally, other cofactors may be required such as proteins generally known as coactivators (also termed adapters or mediators). These proteins do not bind sequence-specifically to DNA and are not involved in basal transcription. They may exert their effect on transcription activation through various mechanisms, including stimulation of DNA-binding of activators, by affecting chromatin structure, or by mediating
- activator-initiation complex interactions 15 activator-initiation complex interactions.
- coactivators include RIP140, TIFl,
- RAP46/Bag-1 RAP46/Bag-1, ARA70, SRC-l/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIBl/RAC3/pCIP as well as the promiscuous coactivator C response element B binding protein, CBP/p300 (for review see Glass et al., Curr. Opin. Cell Biol. 9: 222-232, 1997).
- protein cofactors generally known as corepressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit
- corepressors 20 transcriptional activation in the absence of ligand.
- These corepressors may interact with the unliganded ecdysone receptor to silence the activity at the response element.
- Current evidence suggests that the binding of ligand changes the conformation of the receptor, which results in release of the corepressor and recruitment of the above described coactivators, thereby abolishing their silencing activity.
- corepressors include N-CoR and SMRT (for review, see Horwitz et al. Mol Endocrinol. 10:
- cofactors may either be endogenous within the cell or organism, or may be added exogenously as transgenes to be expressed in either a regulated or unregulated fashion.
- Homodimer complexes of the ecdysone receptor protein, USP, or RXR may also be functional under some circumstances.
- the ecdysone receptor complex typically includes proteins which are members of the nuclear
- DNA binding domain comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response 35 element.
- DBD DNA binding domain
- LBD ligand binding domain
- the term "DNA binding domain” comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response 35 element.
- Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A/B, C, D, E, and in some members F (see US patent 4,981,784 and Evans, Science 240:889-895 (1988)).
- A/B domain corresponds to the transactivation domain
- C corresponds to the DNA binding domain
- D corresponds to the hinge region
- E corresponds to the ligand binding domain.
- Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to "F".
- the DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins.
- This EcR receptor like a subset of the steroid receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Because the domains of EcR, USP, and RXR are modular in nature, the LBD, DBD, and transactivation domains may be interchanged. Gene switch systems are known that incorporate components from the ecdysone receptor complex.
- This two-hybrid system also provides improved sensitivity to non- steroidal ligands for example, diacylhydrazines, when compared to steroidal ligands for example, ponasterone A ("PonA") or muristerone A (“MurA”). That is, when compared to steroids, the non- steroidal ligands provide higher activity at a lower concentration.
- NonA ponasterone A
- MitA muristerone A
- transactivation based on EcR gene switches is often cell-line dependent, it is easier to tailor switching systems to obtain maximum transactivation capability for each application.
- the two-hybrid system avoids some side effects due to overexpression of RXR that often occur when unmodified RXR is used as a switching partner.
- ecdysone receptor in partnership with a dipteran (fruit fly Drosophila melanogaster) or a lepidopteran (spruce bud worm Choristoneurafumiferana) ultraspiracle protein (USP) is constitutively expressed in mammalian ells, while an ecdysone receptor in partnership with a vertebrate retinoid X receptor (RXR) is inducible in mammalian cells (pending application PCT/US01/09050).
- a chimeric RXR ligand binding domain comprising at least two polypeptide fragments, wherein the first polypeptide fragment is from one species of vertebrate/invertebrate RXR and the second polypeptide fragment is from a different species of vertebrate/invertebrate RXR, whereby a vertebrate/invertebrate chimeric RXR ligand binding domain, a vertebrate/vertebrate chimeric RXR ligand binding domain, or an invertebrate/invertebrate chimeric RXR ligand binding domain is produced, can function in an ecdysone receptor-based inducible gene expression system.
- Applicants' novel EcR chimeric RXR-based inducible gene expression system can function similar to or better than both the EcR/vertebrate RXR-based gene expression system (PCT7US01/09050) and the EcR/invertebrate RXR-based gene expression system (US application filed herewith) in terms of ligand sensitivity and magnitude of gene induction.
- the present invention provides an improved EcR-based inducible gene expression system for use in bacterial, fungal, yeast, animal, and mammalian cells.
- This novel gene expression system demonstrates for the first time that a polypeptide comprising a chimeric RXR ligand binding domain can function as a component of an inducible EcR-based inducible gene expression system in yeast and mammalian cells. As discussed herein, this finding is both unexpected and svuprising.
- Applicants' invention relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) an ecdysone receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell, wherein the second gene expression cassette comprises a polynucleotide sequence that encodes a second hybrid polypeptide comprising i) a transactivation domain; and ii) a chimeric retinoid X receptor ligand binding domain.
- the present invention also relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell, wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) a chimeric retinoid X receptor ligand binding domain; and b) a second gene expression cassette that is capable of being expressed in the host cell, wherein the second gene expression cassette comprises a polynucleotide sequence that encodes a second hybrid polypeptide comprising i) a transactivation domain; and ii) an ecdysone receptor ligand binding domain.
- the present invention also relates to a gene expression modulation system according to the present invention further comprising c) a third gene expression cassette comprising: i) a response element to which the DNA-binding domain of the first hybrid polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated.
- the gene whose expression is to be modulated is a homologous gene with respect to the host cell.
- the gene whose expression is to be modulated is a heterologous gene with respect to the host cell.
- the ligands for use in the present invention as described below when combined with an EcR ligand binding domain and a chimeric RXR ligand binding domain, which in turn are bound to the response element linked to a gene, provide the means for external temporal regulation of expression of the gene.
- the binding mechanism or the order in which the various components of this invention bind to each other that is, for example, ligand to receptor, first hybrid polypeptide to response element, second hybrid polypeptide to promoter, etc., is not critical. Binding of the ligand to the EcR ligand binding domain and the chimeric RXR ligand binding domain enables expression or suppression of the gene.
- This mechanism does not exclude the potential for ligand binding to EcR or chimeric RXR, and the resulting fo ⁇ nation of active homodimer complexes (e.g. EcR + EcR or chimeric RXR + chimeric RXR).
- one or more of the receptor domains is varied producing a hybrid gene switch.
- one or more of the three domains, DBD, LBD, and transactivation domain may be chosen from a source different than the source of the other domains so that the hybrid genes and the resulting hybrid proteins are optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element, i addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski, et al.
- Promoters maybe constitutively or inducibly regulated or may be tissue-specific (that is, expressed only in a particular type of cells) or specific to certain developmental stages of the organism.
- the novel EcR/chimeric RXR-based inducible gene expression system of the invention comprises gene expression cassettes that are capable of being expressed in a host cell, wherein the gene expression cassettes each comprise a polynucleotide encoding a hybrid polypeptide.
- Applicants' invention also provides gene expression cassettes for use in the gene expression system of the invention.
- the present invention provides a gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide.
- the present invention provides a gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide that encodes a hybrid polypeptide comprising either i) a DNA-binding domain that recognizes a response element, or ii) a transactivation domain; and an ecdysone receptor ligand binding domain or a chimeric retinoid X receptor ligand binding domain.
- the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain that recognizes a response element and an EcR ligand binding domain.
- the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain that recognizes a response element and a chimeric RXR ligand binding domain.
- the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain and an EcR ligand binding domain.
- the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain and a chimeric RXR ligand binding domain.
- the ligand binding domain (LBD) is an EcR LBD, a chimeric RXR
- the LBD is an EcR LBD or a chimeric RXR LBD.
- the LBD is from a truncated EcR LBD or a truncated chimeric RXR LBD.
- a truncation mutation may be made by any method used in the art, including but not limited to restriction endonuclease digestion/deletion, PCR-mediated oligonucleotide- directed deletion, chemical mutagenesis, DNA strand breakage, and the like.
- the EcR may be an invertebrate EcR, preferably selected from the class Arthropod.
- the EcR is selected from the group consisting of a Lepidopteran EcR, a Dipteran EcR, an Orthopteran EcR, a Homopteran EcR and a Hemipteran EcR. More preferably, the EcR for use is a spruce budwonn Choristoneurafumiferana EcR ("CfEcR”), a beetle Tenebrio molitor EcR (“TmEcR”), a Manduca sexta
- EcR a Heliothies virescens EcR (“HvEcR”), a midge Chironomus tentans EcR (“CfEcR”), a silk moth Bombyx tnori EcR (“BmEcR”), a fruit fly Drosophila melanogaster EcR (“DmEcR”), a mosquito Aedes aegypti EcR (“AaEcR”), a blowfly Lucilia capitata EcR (“LcEcR”), a blowfly Lucilia cuprina EcR (“LucEcR”), a Mediterranean fruit fly Ceratitis capitata EcR (“CcEcR”), a locust Locusta migratoria EcR (“LmEcR”), an aphid Myzus persicae EcR (“MpEcR”), a fiddler crab Celuca pugilator EcR (“CpEcR”), an ixodid tic
- the EcR LBD comprises full-length EF domains.
- the-full length EF domains are encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
- the LBD is from a truncated EcR LBD.
- the EcR LBD truncation results in a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 amino acids.
- the EcR LBD truncation result in a deletion of at least a partial polypeptide domain.
- the EcR LBD truncation results in a deletion of at least an entire polypeptide domain. More preferably, the EcR polypeptide truncation results in a deletion of at least an A/B-domain, a C- domain, a D-domain, an F-domain, an A B/C-domains, an A/B/l/2-C-domains, an A/B/C/D-domains, an A B/C/D/F-domains, an A/B/F-domains, an A/B/C/F-domains, a partial E-domain, or a partial F-domain.
- a combination of several partial and/or complete domain deletions may also be performed.
- the ecdysone receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 (CfEcR-EF), SEQ ID NO: 2 (DmEcR-EF), SEQ ID NO: 3 (CfEcR-DE), and SEQ ID NO: 4 (DmEcR-DE).
- the ecdysone receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ED NO: 65 (CfEcR-DEF), SEQ ID NO: 59 (CfEcR-CDEF), SEQ ID NO: 67 (DmEcR-DEF), SEQ ID NO: 71 (TmEcR-DEF) and SEQ ID NO: 73 (AmaEcR-DEF).
- the ecdysone receptor ligand binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (CfEcR-EF), SEQ ID NO: 6 (DmEcR-EF), SEQ ID NO: 7 (CfEcR-DE), and SEQ ID NO: 8 (DmEcR-DE).
- the ecdysone receptor ligand binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 70 (CfEcR-CDEF), SEQ ID NO: 58 (DmEcR-DEF), SEQ ID NO: 72 (TmEcR-DEF), and SEQ ID NO: 74 (AmaEcR-DEF).
- the chimeric RXR ligand binding domain comprises at least two polypeptide fragments selected from the group consisting of a vertebrate species RXR polypeptide fragment, an invertebrate species RXR polypeptide fragment, and a non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment.
- a chimeric RXR ligand binding domain according to the invention may comprise at least two different species RXR polypeptide fragments, or when the species is the same, the two or more polypeptide fragments may be from two or more different isoforms of the species RXR polypeptide fragment.
- the vertebrate species RXR polypeptide fragment is from a mouse Mus musculus RXR ("MmRXR") or a human Homo sapiens RXR ("HsRXR").
- the RXR polypeptide may be an RXR « , RXR ⁇ , or RXR ⁇ isoform.
- the vertebrate species RXR polypeptide fragment is from a vertebrate species RXR-EF domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.
- the vertebrate species RXR polypeptide fragment is from a vertebrate species RXR-EF domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
- the invertebrate species RXR polypeptide fragment is from a locust Locusta migratoria ultraspiracle polypeptide ("LmUSP”), an ixodid tick Amblyomma americanum RXR homolog 1 (“AmaRXRl”), a ixodid tick Amblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), and an aphid Myzus persicae RXR homolog (“MpRXR”).
- LmUSP locust Locusta migratoria ultraspiracle polypeptide
- AmaRXRl ixodid tick Amblyomma americanum RXR homolog 1
- the invertebrate species RXR polypeptide fragment is from a invertebrate species RXR-EF domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ED NO: 26.
- the invertebrate species RXR polypeptide fragment is from a invertebrate species RXR-EF domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.
- the invertebrate species RXR polypeptide fragment is from a non-Dipteran/non-Lepidopteran invertebrate species RXR homolog.
- the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one invertebrate species RXR polypeptide fragment.
- the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment.
- the chimeric RXR ligand binding domain comprises at least one invertebrate species RXR polypeptide fragment and one non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment.
- the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one different vertebrate species RXR polypeptide fragment.
- the chimeric RXR ligand binding domain comprises at least one invertebrate species RXR polypeptide fragment and one different invertebrate species RXR polypeptide fragment.
- the chimeric RXR ligand binding domain comprises at least one non-Dipteran/non-Lepidopteran invertebrate species RXR polypeptide fragment and one different non-Dipteran non-Lepidopteran invertebrate species RXR polypeptide fragment.
- the chimeric RXR LBD comprises an RXR LBD domain comprising at least one polypeptide fragment selected from the group consisting of an EF-domain helix 1, an EF- domain helix 2, an EF-domain helix 3, an EF-domain helix 4, an EF-domain helix 5, an EF-domain helix 6, an EF-domain helix 7, an EF-domain helix 8, and EF-domain helix 9, an EF-domain helix 10, an EF- domain helix 11, an EF-domain helix 12, an F-domain, and an EF-domain ⁇ -pleated sheet, wherein the polypeptide fragment is from a different species RXR, i.e., chimeric to the RXR LBD domain, than the RXR LBD domain.
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-6, helices 1-7, helices 1-8, helices 1-9, helices 1-10, helices 1-11, or helices 1-12 of a first species RXR according to the invention
- the second polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 7-12, helices 8-12, helices 9-12, helices 10- 12, helices 11-12, helix 12, or F domain of a second species RXR according to the invention, respectively.
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-6 of a first species RXR according to the invention
- the second polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 7-12 of a second species RXR according to the invention.
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-7 of a first species RXR according to the invention
- the second polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 8-12 of a second species RXR according to the invention.
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-8 of a first species RXR according to the invention
- the second polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 9-12 of a second species RXR according to the invention
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-9 of a first species RXR according to the invention
- the second polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 10-12 of a second species RXR according to the invention.
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-10 of a first species RXR according to the invention, and the second polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 11-12 of a second species RXR according to the invention.
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-11 of a first species RXR according to the invention
- the second polypeptide fragment of the chimeric RXR ligand binding domain comprises helix 12 of a second species RXR according to the invention.
- the first polypeptide fragment of the chimeric RXR ligand binding domain comprises helices 1-12 of a first species RXR according to the invention
- the second polypeptide fragment of the chimeric RXR ligand binding domain comprises an F domain of a second species RXR according to the invention.
- the LBD is from a truncated chimeric RXR ligand binding domain.
- the chimeric RXR LBD truncation results in a deletion of at least 1, 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 amino acids.
- the chimeric RXR LBD truncation results in a deletion of at least a partial polypeptide domain. More preferably, the chimeric RXR LBD truncation results in a deletion of at least an entire polypeptide domain.
- the cliimeric RXR LBD truncation results in a deletion of at least a partial E-domain, a complete E-domain, a partial F-domain, a complete F-domain, an EF-domain helix 1, an EF-domain helix 2, an EF-domain helix 3, an EF-domain helix 4, an EF-domain helix 5, an EF-domain helix 6, an EF-domain helix 7, an EF-domain helix 8, and EF-domain helix 9, an EF-domain helix 10, an EF-domain helix 11, an EF-domain helix 12, or an EF-domain ⁇ -pleated sheet.
- a combination of several partial and or complete domain deletions may also be performed.
- the truncated chimeric RXR ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.
- the truncated chimeric RXR ligand binding domain comprises a nucleic acid sequence of SEQ ID NO: 39, SEQ ID NO:
- the chimeric RXR ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a) SEQ ID NO: 45, b) nucleotides 1-348 of SEQ BD NO: 13 and nucleotides 268-630 of SEQ ID NO: 21, c) nucleotides 1-408 of SEQ ID NO: 13 and nucleotides 337-630 of SEQ ID NO: 21, d) nucleotides 1-465 of SEQ ID
- the chimeric RXR ligand binding domain comprises an amino acid sequence selected from the group consisting of a) SEQ ID NO: 46, b) amino acids 1-116 of SEQ ID NO: 13 and amino acids 90-210 of SEQ ID NO: 21, c) amino acids 1-136 of SEQ ID NO: 13 and amino acids 113-210 of SEQ ID NO: 21, d) amino acids 1-155 of SEQ ID NO: 13 and amino acids 135-210 of SEQ ID NO: 21, e) amino acids 1-185 of SEQ ID NO: 13 and amino acids 164-210 of SEQ ID NO: 21, f) amino acids 1-208 of SEQ ID NO: 13 and amino acids 183-210 of SEQ ID NO: 21, g) amino acids 1-215 of SEQ ID NO: 13 and amino acids 201-210 of SEQ ID NO: 21, and h) amino acids 1-239 of SEQ ID 5 NO: 13 and amino acids 205-210 of SEQ ID NO: 21.
- EcR, vertebrate RXR, invertebrate RXR, and chimeric RXR also include synthetic and hybrid EcR, vertebrate RXR, invertebrate RXR, and chimeric RXR, and their homologs.
- the DNA binding domain can be any DNA binding domain with a known response element
- the DBD is a GAL4 DBD, a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, or a yeast put DBD. More preferably, the DBD is a GAL4 DBD [SEQ ID NO: 47 (polynucleotide) or SEQ ID NO: 48 (polypeptide)] or a LexA DBD [(SEQ ID NO: 49 (polynucleotide) or SEQ ID NO: 50 (polypeptide)].
- the transactivation domain may be any steroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acid AD, a VP16 AD, a GAL4 AD, an NF- ⁇ B AD, a BP64 AD, a B42 acidic activation domain (B42AD), or an analog, combination, or modification thereof.
- the AD is a synthetic or cliimeric AD, or is obtained from a VP16, GAL4, NF-kB, or B42 acidic activation domain AD.
- the AD is a VP16 AD [SEQ ID NO: 51 (polynucleotide) or SEQ ID NO: 52 (polypeptide)] or a B42 AD [SEQ ID NO: 53 (polynucleotide) or SEQ ID NO: 54 (polypeptide)].
- the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 47) and a LexA DBD (SEQ ID NO:
- EcR ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 65 (CfEcR-DEF), SEQ ID NO: 59 (CfEcR-CDEF), SEQ ID NO: 67 (DmEcR-DEF), SEQ ID NO: 71 (TmEcR-DEF) and SEQ ID NO: 73 (AmaEcR-DEF).
- the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group
- EcR ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 70 (CfEcR-CDEF), SEQ ID NO: 58 (DmEcR-DEF), SEQ ID NO: 72 (TmEcR-DEF), and SEQ ID NO: 74 (AmaEcR-DEF).
- the gene expression cassette encodes a hybrid polypeptide 35 comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 47) and a LexA DBD (SEQ ID NO: 49), and a chimeric RXR ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a) SEQ ID NO: 45, b) nucleotides 1-348 of SEQ ID NO: 13 and nucleotides 268-630 of SEQ ID NO: 21, c) nucleotides 1-408 of SEQ ID NO: 13 and nucleotides 337-630 of SEQ ID NO: 21, d) nucleotides 1-465 of SEQ ID NO: 13 and nucleotides 403-630 of SEQ ID NO: 21, e) nucleotides 1-555 of SEQ
- the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 48) and a LexA DBD (SEQ ID NO: 50), and a chimeric RXR ligand binding domain comprising an amino acid sequence selected from the group consisting of a) SEQ ID NO: 46, b) amino acids 1-116 of SEQ ID NO: 13 and amino acids 90-210 of SEQ ID NO: 21, c) amino acids 1-136 of SEQ ID NO: 13 and amino acids 113-210 of SEQ ID NO: 21, d) amino acids 1-155 of SEQ ID NO: 13 and amino acids 135-210 of SEQ ID NO: 21, e) amino acids 1-185 of SEQ ID NO: 13 and amino acids 164-210 of SEQ ID NO: 21, f) amino acids 1-208 of SEQ ID NO: 13 and amino acids 183-210 of SEQ ID NO: 21, g) amino acids 1-215 of SEQ ID NO: 46,
- the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 51 or SEQ ID NO: 53, and an EcR ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 65 (CfEcR-DEF), SEQ ID NO: 59 (CfEcR-CDEF), SEQ ID NO: 67 (DmEcR-DEF), SEQ ID NO: 71 (TmEcR-DEF) and SEQ ID NO: 73 (AmaEcR-DEF).
- the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence of SEQ ID NO: 52 or SEQ ID NO:
- EcR ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 70 (CfEcR-CDEF), SEQ ID NO: 58 (DmEcR- DEF), SEQ ED NO: 72 (TmEcR-DEF), and SEQ ID NO: 74 (AmaEcR-DEF).
- the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of
- the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence of SEQ ED NO: 52 or SEQ ED NO: 54 and a chimeric RXR ligand binding domain comprising an amino acid sequence selected from the group consisting of a) SEQ ID NO: 46, b) amino acids 1-116 of SEQ ID NO: 13 and amino acids 90- 210 of SEQ ID NO: 21, c) amino acids 1-136 of SEQ ID NO: 13 and amino acids 113-210 of SEQ ID NO: 21, d) amino acids 1-155 of SEQ ID NO: 13 and amino acids 135-210 of SEQ ID NO: 21, e) amino acids 1-185 of SEQ ID NO: 13 and amino acids 164-210 of SEQ ID NO: 21, f) amino acids 1-208 of SEQ ID NO: 13 and amino acids 183-210 of SEQ ID NO: 21, g) amino acids 1-215 of SEQ ID NO: 13 and amino acids 201-210 of SEQ ED NO: 21, and h)
- the response element may be any response element with a known DNA binding domain, or an analog, combination, or modification thereof.
- a single RE may be employed or multiple REs, either multiple copies of the same RE or two or more different REs, may be used in the present invention.
- the RE is an RE from GAL4 ("GAL4RE"), LexA, a steroid/thyroid hormone nuclear receptor RE, or a synthetic RE that recognizes a synthetic DNA binding domain.
- GAL4RE GAL4
- LexA a steroid/thyroid hormone nuclear receptor RE
- the RE is a GAL4RE comprising a polynucleotide sequence of SEQ ID NO: 55 or a LexARE (operon "op") comprising a polynucleotide sequence of SEQ ID NO: 56 (2XLexAop).
- the first hybrid protein is substantially free of a transactivation domain and the second hybrid protein is substantially free of a DNA binding domain.
- substantially free means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.
- the present invention also relates to a gene expression cassette comprising: i) a response element comprising a domain to which a polypeptide comprising a DNA binding domain binds; ii) a promoter that is activated by a polypeptide comprising a transactivation domain; and iii) a gene whose expression is to be modulated.
- Genes of interest for use in Applicants' gene expression cassettes may be endogenous genes or heterologous genes.
- Nucleic acid or amino acid sequence information for a desired gene or protein can be located in one of many public access databases, for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology related journal publications. Thus, those skilled in the art have access to nucleic acid sequence information for virtually all known genes. Such information can then be used to construct the desired constructs for the insertion of the gene of interest within the gene expression cassettes used in Applicants' methods described herein.
- genes of interest for use in Applicants' gene expression cassettes include, but are not limited to: genes encoding therapeutically desirable polypeptides or products that may be used to treat a condition, a disease, a disorder, a dysfunction, a genetic defect, such as monoclonal antibodies, enzymes, proteases, cytokines, interferons, insulin, erthropoietin, clotting factors, other blood factors or components, viral vectors for gene therapy, virus for vaccines, targets for drug discovery, functional genomics, and proteomics analyses and applications, and the like.
- the novel ecdysone receptor/chimeric retinoid X receptor-based inducible gene expression system of the invention comprises a gene expression cassette comprising a polynucleotide that encodes a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain, and b) an EcR ligand binding domain or a chimeric RXR ligand binding domain.
- a gene expression cassette comprising a polynucleotide that encodes a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain, and b) an EcR ligand binding domain or a chimeric RXR ligand binding domain.
- EcR-based gene expression system to modulate the expression of a gene within a host cell.
- the present invention provides an isolated polynucleotide that encodes a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain according to the invention, and b) an EcR ligand binding domain or a chimeric RXR ligand binding domain according to the invention.
- the present invention also relates to an isolated polynucleotide that encodes a cliimeric RXR ligand binding domain according to the invention.
- the present invention also relates to an isolated polynucleotide that encodes a truncated EcR LBD or a truncated chimeric RXR LBD comprising a truncation mutation according to the invention. Specifically, the present invention relates to an isolated polynucleotide encoding a truncated EcR or
- chimeric RXR ligand binding domain comprising a truncation mutation that affects ligand binding activity or ligand sensitivity that is useful in modulating gene expression in a host cell.
- the isolated polynucleotide encoding an EcR LBD comprises a polynucleotide sequence selected from the group consisting of SEQ ED NO: 65 (CfEcR-DEF), SEQ ED NO: 59 (CfEcR-CDEF), SEQ ID NO: 67 (DmEcR-DEF), SEQ ED NO: 71 (TmEcR-DEF) and SEQ ID 25 NO: 73 (AmaEcR-DEF).
- the isolated polynucleotide encodes an EcR LBD comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 57 (CfEcR-DEF), SEQ ID NO: 70 (CfEcR-CDEF), SEQ ID NO: 58 (DmEcR-DEF), SEQ ID NO: 72 (TmEcR-DEF), and SEQ ED NO: 74 (AmaEcR-DEF).
- the isolated polynucleotide encoding a chimeric RXR LBD comprises a polynucleotide sequence selected from the group consisting of a) SEQ ID NO: 45, b) nucleotides 1-348 of SEQ ED NO: 13 and nucleotides 268-630 of SEQ ID NO: 21, c) nucleotides 1-408 of SEQ ID NO: 13 and nucleotides 337-630 of SEQ ID NO: 21, d) nucleotides 1-465 of SEQ ID NO: 13 and nucleotides 403-630 of SEQ ID NO: 21, e) nucleotides 1-555 of SEQ ID NO: 13 and nucleotides 35 490-630 of SEQ ID NO: 21, f) nucleotides 1-624 of SEQ ID NO: 13 and nucleotides 547-630 of SEQ ID NO: 21, g) nucleotides 1-645 of SEQ ED NO: 13 and nucleotides
- the isolated polynucleotide encodes a chimeric RXR LBD comprising an amino acid sequence consisting of a) SEQ ED NO: 46, b) amino acids 1-116 of SEQ ID NO: 13 and amino acids 90-210 of SEQ ID NO: 21, c) amino acids 1-136 of SEQ ID NO: 13 and amino acids 113-210 of SEQ ED NO: 21, d) amino acids 1-155 of SEQ ED NO: 13 and amino acids 135-210 of SEQ ID NO: 21, e) amino acids 1-185 of SEQ ID NO: 13 and amino acids 164-210 of SEQ ID NO: 21, f) amino acids 1-208 of SEQ ID NO: 13 and amino acids 183-210 of SEQ ID NO: 21, g) amino acids 1-215 of SEQ ID NO: 13 and amino acids 201-210 of SEQ ID NO: 21, and h) amino acids 1-239 of SEQ ID NO: 13 and amino acids 205-210 of SEQ ID NO: 21.
- the present invention relates to an isolated polynucleotide encoding a truncated chimeric RXR LBD comprising a truncation mutation, wherein the mutation reduces ligand binding activity or ligand sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated polynucleotide encoding a truncated chimeric RXR LBD comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated polynucleotide encoding a truncated chimeric RXR LBD comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention also relates to an isolated polynucleotide encoding a truncated chimeric RXR LBD comprising a truncation mutation, wherein the mutation enhances ligand binding activity or ligand sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated polynucleotide encoding a truncated chimeric RXR LBD comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated polynucleotide encoding a truncated chimeric RXR LBD comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention also relates to an isolated polynucleotide encoding a truncated chimeric retinoid X receptor LBD comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated chimeric retinoid X receptor LBD and a dimeriz-ation partner.
- the dimerization partner is an ecdysone receptor polypeptide.
- the dimerization partner is a truncated EcR polypeptide. More preferably, the dimerization partner is an EcR polypeptide in which domains A/B have been deleted.
- the dimerization partner is an EcR polypeptide comprising an amino acid sequence of SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 58 (DmEcR-DEF), SEQ ID NO: 70 (CfEcR-CDEF), SEQ ID NO: 72 (TmEcR-DEF) or SEQ ID NO: 74 (AmaEcR-DEF).
- EcR polypeptide comprising an amino acid sequence of SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 58 (DmEcR-DEF), SEQ ID NO: 70 (CfEcR-CDEF), SEQ ID NO: 72 (TmEcR-DEF) or SEQ ID NO: 74 (AmaEcR-DEF).
- the novel ecdysone receptor/chimeric retinoid X receptor-based inducible gene expression system of the invention comprises a gene expression cassette comprising a polynucleotide that encodes a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain, and b) an EcR ligand binding domain or a chimeric RXR ligand binding domain.
- These gene expression cassettes, the polynucleotides they comprise, and the hybrid polypeptides they encode are useful as components of an EcR/chimeric RXR-based gene expression system to modulate the expression of a gene within a host cell.
- the present invention also relates to a hybrid polypeptide comprising a) a DNA binding domain or a transactivation domain according to the invention, and b) an EcR ligand binding domain or a chimeric RXR ligand binding domain according to the invention.
- the present invention also relates to an isolated polypeptide comprising a chimeric RXR ligand binding domain according to the invention.
- the present invention also relates to an isolated truncated EcR LBD or an isolated truncated chimeric RXR LBD comprising a truncation mutation according to the invention. Specifically, the present invention relates to an isolated truncated EcR LBD or an isolated truncated chimeric RXR LBD comprising a truncation mutation that affects ligand binding activity or ligand sensitivity.
- the isolated EcR LBD polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO: 65 (CfEcR- DEF), SEQ BD NO: 59 (CfEcR-CDEF), SEQ BD NO: 67 (DmEcR-DEF), SEQ ID NO: 71 (TmEcR-DEF) and SEQ ID NO: 73 (AmaEcR-DEF).
- a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO: 65 (CfEcR- DEF), SEQ BD NO: 59 (CfEcR-CDEF), SEQ BD NO: 67 (DmEcR-DEF), SEQ ID NO: 71 (TmEcR-DEF) and SEQ ID NO: 73 (AmaEcR-DEF).
- the isolated EcR LBD polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 70 (CfEcR- CDEF), SEQ ID NO: 58 (DmEcR-DEF), SEQ ID NO: 72 (TmEcR-DEF), and SEQ ID NO: 74 (AmaEcR- DEF).
- the isolated truncated chimeric RXR LBD is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) SEQ ID NO: 45, b) nucleotides 1-348 of SEQ ID NO: 13 and nucleotides 268-630 of SEQ ID NO: 21, c) nucleotides 1-408 of SEQ ED NO: 13 and nucleotides 337-630 of SEQ ED NO: 21, d) nucleotides 1-465 of SEQ ED NO: 13 and nucleotides 403-630 of SEQ ID NO: 21, e) nucleotides 1-555 of SEQ D NO: 13 and nucleotides 490-630 of SEQ ED NO: 21, f) nucleotides 1-624 of SEQ ED NO: 13 and nucleotides 547-630 of SEQ ED NO: 21, g) nucleotides 1-645 of SEQEQ ID NO: 45
- the isolated truncated chimeric RXR LBD comprises an amino acid sequence selected from the group consisting of a) SEQ ED NO: 46, b) amino acids 1-116 of SEQ ED NO: 13 and amino acids 90-210 of SEQ ED NO: 21, c) amino acids 1-136 of SEQ ED NO: 13 and amino acids 113-210 of SEQ ED NO: 21, d) amino acids 1-155 of SEQ ED NO: 13 and amino acids 135-210 of SEQ ED NO: 21, e) amino acids 1-185 of SEQ ED NO: 13 and amino acids 164-210 of SEQ ID NO: 21, f) amino acids 1-208 of SEQ ED NO: 13 and amino acids 183-210 of SEQ ED NO: 21, g) amino acids 1-215 of SEQ ED NO: 13 and amino acids 201-210 of SEQ ED NO: 21, and h) amino acids 1-239 of SEQ ED NO: 13 and amino acids 205-210 of SEQ ED NO: 21.
- the present invention comprises an amino
- the present invention relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that reduces ligand binding activity or ligand sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated truncated cliimeric RXR LBD comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention relates to an isolated truncated chimeric
- RXR LBD comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the truncated chimeric RXR LBD.
- the present invention also relates to an isolated truncated chimeric RXR LBD comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated chimeric RXR LBD and a dimerization partner.
- the dimerization partner is an ecdysone receptor polypeptide.
- the dimerization partner is a truncated EcR polypeptide.
- the dimerization partner is an EcR polypeptide in which domains A/B or A/B/C have been deleted.
- the dimerization partner is an EcR polypeptide comprising an amino acid sequence of SEQ BD NO: 57 (CfEcR-DEF), SEQ ED NO: 58 (DmEcR-DEF), SEQ BD NO: 70 (CfEcR-CDEF), SEQ ID NO: 72 (TmEcR-DEF) or SEQ ID NO: 74 (AmaEcR-DEF).
- Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention.
- Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising: i) a response element comprising a domain recognized by the DNA binding domain of the first hybrid polypeptide; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated, whereby upon introduction of the ligand into the host cell, expression of the gene is modulated.
- the invention also provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; b) introducing into the host cell a gene expression cassette comprising i) a response element comprising a domain recognized by the DNA binding domain from the first hybrid polypeptide; ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and iii) a gene whose expression is to be modulated; and c) introducing into the host cell a ligand; whereby expression of the gene is modulated in the host cell.
- Genes of interest for expression in a host cell using Applicants' methods may be endogenous genes or heterologous genes.
- Nucleic acid or amino acid sequence information for a desired gene or protein can be located in one of many public access databases, for example, GENBANK, EMBL, Swiss- Prot, and PIR, or in many biology related journal publications. Thus, those skilled in the art have access to nucleic acid sequence information for virtually all known genes. Such information can then be used to construct the desired constructs for the insertion of the gene of interest within the gene expression cassettes used in Applicants' methods described herein.
- genes of interest for expression in a host cell using Applicants' methods include, but are not limited to: genes encoding therapeutically desirable polypeptides or products that may be used to treat a condition, a disease, a disorder, a dysfunction, a genetic defect, such as monoclonal antibodies, enzymes, proteases, cytokines, interferons, insulin, erthropoietin, clotting factors, other blood factors or components, viral vectors for gene therapy, virus for vaccines, targets for drug discovery, functional genomics, and proteomics analyses and applications, and the like.
- Acceptable ligands are any that modulate expression of the gene when binding of the DNA binding domain of the two-hybrid system to the response element in the presence of the ligand results in activation or suppression of expression of the genes.
- Preferred ligands include ponasterone, muristerone A, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazines such as those disclosed in U. S. Patents No. 6,013,836; 5,117,057; 5,530,028; and 5,378,726; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No.
- N-alkyl-N,N'-diaroylhydrazines such as those disclosed in U. S. Patent No. 5,225,443
- N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No. 234,994
- N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U. S. Patent No. 4,985,461; each ofwhich is inco ⁇ orated herein by reference and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylha ⁇ agide, and the like.
- the ligand for use in Applicants' method of modulating expression of gene is a compound of the formula:
- E is a (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;
- R 2 is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHC1 2 , CH 2 F, CH 2 C1, CH 2 OH, CH 2 OMe, CH 2
- R 4 , R 5 , and R 6 are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHC1 2 , CH 2 F, CH 2 C1, CH 2 OH, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.
- a second ligand may be used in addition to the first ligand discussed above in Applicants' method of modulating expression of a gene, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of retinoic acid.
- the present invention provides for modulation of gene expression in prokaryotic and eukaryotic host cells.
- the present invention also relates to a method for modulating gene expression in a host cell selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell.
- the host cell is a yeast cell, a hamster cell, a mouse cell, a monkey cell, or a human cell.
- Expression in transgenic host cells may be useful for the expression of various polypeptides of interest including but not limited to therapeutic polypeptides, pathway intermediates; for the modulation of pathways already existing in the host for the synthesis of new products heretofore not possible using the host; cell based assays; functional genomics assays, biotherapeutic protein production, proteomics assays, and the like. Additionally the gene products may be useful for conferring higher growth yields of the host or for enabling an alternative growth mode to be utilized.
- the gene expression modulation system of the present invention may be used to modulate gene expression in a host cell.
- Expression in transgenic host cells may be useful for the expression of various genes of interest.
- Applicants' invention provides an isolated host cell comprising a gene expression system according to the invention.
- the present invention also provides an isolated host cell comprising a gene expression cassette according to the invention.
- Applicants' invention also provides an isolated host cell comprising a polynucleotide or a polypeptide according to the invention.
- the isolated host cell may be either a prokaryotic or a eukaryotic host cell.
- the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell.
- preferred host cells include, but are not limited to, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as those in the genera Synechocystis, Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella, animal, and mammalian host cells.
- the host cell is a yeast cell selected from the group consisting of a yeast cell selected from the group consisting of a yeast cell selected from the group consisting of
- Saccharomyces Saccharomyces, a Pichia, and a Candida host cell.
- the host cell is a hamster cell.
- the host cell is a murine cell.
- the host cell is a monkey cell.
- the host cell is a human cell.
- Host cell transformation is well known in the art and may be achieved by a variety of methods including but not limited to electroporation, viral infection, plasmid/vector transfection, non-viral vector mediated transfection, particle bombardment, and the like.
- Expression of desired gene products involves culturing the transformed host cells under suitable conditions and inducmg expression of the transformed gene.
- Culture conditions and gene expression protocols in prokaryotic and eukaryotic cells are well known in the art (see General Methods section of Examples). Cells may be harvested and the gene products isolated according to protocols specific for the gene product.
- a host cell may be chosen which modulates the expression of the inserted polynucleotide, or modifies and processes the polypeptide product in the specific fashion desired.
- Different host cells have characteristic and specific mechanisms for the translational and post- translational processing and modification [e.g., glycosylation, cleavage (e.g., of signal sequence)] of proteins.
- Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
- expression in a bacterial system can be used to produce a non-glycosylated core protein product.
- a polypeptide expressed in bacteria may not be properly folded.
- Expression in yeast can produce a glycosylated product.
- Expression in eukaryotic cells can increase the likelihood of "native" glycosylation and folding of a heterologous protein.
- expression in mammalian cells can provide a tool for reconstituting, or constituting, the polypeptide' s activity.
- different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.
- Applicants' invention also relates to a non-human organism comprising an isolated host cell according to the invention.
- the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, an animal, and a mammal. More preferably, the non-human organism is a yeast, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, or a chimpanzee.
- the non-human organism is a yeast selected from the group consisting oi Saccharomyces, Pichia, and Candida. In another specific embodiment, the non-human organism is a Mus musculus mouse.
- RNA preferably mRNA species.
- Such measurements are conveniently conducted by measuring cDNA abundances by any of several existing gene expression technologies.
- Nucleic acid array technology is a useful technique for determining differential mRNA expression.
- Such technology includes, for example, oligonucleotide chips and DNA microarrays. These techniques rely on DNA fragments or oligonucleotides which correspond to different genes or cDNAs which are immobilized on a solid support and hybridized to probes prepared from total mRNA pools extracted from cells, tissues, or whole organisms and converted to cDNA.
- Oligonucleotide chips are arrays of oligonucleotides synthesized on a substrate using photolithographic techniques. Chips have been produced which can analyze for up to 1700 genes.
- DNA microarrays are arrays of DNA samples, typically PCR products, that are robotically printed onto a microscope slide.
- oligonucleotide chips typically utilize 25-mer oligonucleotides which allow fractionation of short DNA molecules whereas the larger DNA targets of microarrays, approximately 1000 base pairs, may provide more sensitivity in fractionating complex DNA mixtures.
- Another useful measurement of Applicants ' methods of the invention is that of determining the translation state of the cell by measuring the abundances of the constituent protein species present in the cell using processes well known in the art.
- an assay may be employed in which changes in such functions as cell growth, apoptosis, senescence, differentiation, adhesion, binding to a specific molecules, binding to another cell, cellular organization, organogenesis, intracellular transport, transport facilitation, energy conversion, metabolism, myogenesis, neurogenesis, and/or hematopoiesis is measured.
- selectable marker or reporter gene expression may be used to measure gene expression modulation using Applicants' invention.
- labeled proteins can be used to detect a particular nucleic acid sequence to which it hybidizes. i some cases it is necessary to amplify the amount of a nucleic acid sequence.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- SDA strand displacement amplification
- transcription-based amplification and the like.
- PCR is carried out in accordance with known techniques in which, for example, a nucleic acid sample is treated in the presence of a heat stable DNA polymerase, under hybridizing conditions, with one pair of oligonucleotide primers, with one primer hybridizing to one strand (template) of the specific sequence to be detected.
- the primers are sufficiently complementary to each template strand of the specific sequence to hybridize therewith.
- An extension product of each primer is synthesized and is complementary to the nucleic acid template strand to which it hybridized.
- the extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.
- the sample may be analyzed as described above to assess whether the sequence or sequences to be detected are present.
- the present invention may be better understood by reference to the following non-limiting
- Manipulations of genetic sequences maybe accomplished using the suite of programs available from the Genetics Computer Group Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI). Where the GCG program "Pileup” is used the gap creation default value of 12, and the gap extension default value of 4 may be used. Where the CGC “Gap” or “Bestfit” program is used the default gap creation penalty of 50 and the default gap extension penalty of 3 may be used. In any case where GCG program parameters are not prompted for, in these or any other GCG program, default values may be used.
- EcR/chimeric RXR-based inducible gene expression modulation system is useful in various applications including gene tlierapy, expression of proteins of interest in host cells, production of transgenic organisms, and cell-based assays.
- Applicants have made the su ⁇ rising discovery that a chimeric retinoid X receptor ligand binding domain can substitute for either parent RXR polypeptide and function inducibly in an EcR/chimeric RXR- based gene expression modulation system upon binding of ligand.
- the chimeric RXR polypeptide may also function better than either parent/donor RXR ligand binding domain.
- EcR-based gene expression cassettes based on the spruce budworm Choristoneurafumiferana EcR ("CfEcR”), C.fumiferana ultraspiracle (“CfUSP”), Drosophila melanogaster EcR (“DmEcR”), D. melanogaster USP (“DmUSP”), Tenebrio molitor EcR (“TmEcR”), Amblyomma americanum EcR(" AmaEcR”), A. americanum RXR homolog 1 (“AmaRXRl”), A.
- AmaRXR2 mouse Mus musculus retinoid X receptor ⁇ isoform
- MmRXR ⁇ mouse Mus musculus retinoid X receptor ⁇ isoform
- HsRXR ⁇ human Homo sapiens retinoid X receptor ⁇ isoform
- Locusta migratoria ultraspiracle LmUSP
- the prepared receptor constructs comprise 1) an EcR ligand binding domain (LBD), a vertebrate RXR (MmRXR ⁇ or HsRXR ⁇ ) LBD, an invertebrate USP (CfUSP or DmUSP) LBD, an invertebrate RXR (LmUSP, AmaRXRl or AmaRXR2) LBD, or a chimeric RXR LBD comprising a vertebrate RXR LBD fragment and an invertebrate RXR LBD fragment; and 2) a GAL4 or LexA DNA binding domain (DBD) or a VP 16 or B42 acidic activator transactivation domain (AD).
- LBD EcR ligand binding domain
- MmRXR ⁇ or HsRXR ⁇ vertebrate RXR
- CfUSP or DmUSP invertebrate USP
- LmUSP invertebrate RXR
- LmUSP invertebrate RXR
- the reporter constructs include a reporter gene, luciferase or LacZ, operably linked to a synthetic promoter construct that comprises either a GAL4 response element or a LexA response element to which the Gal4 DBD or LexA DBD binds, respectively.
- a synthetic promoter construct that comprises either a GAL4 response element or a LexA response element to which the Gal4 DBD or LexA DBD binds, respectively.
- Various combinations of these receptor and reporter constructs were cotransfected into mammalian cells as described in Examples 2-6 infra.
- Ecdysone receptor-based gene expression cassette pairs were constructed as followed, using standard cloning methods available in the art. The following is brief description of preparation and composition of each switch used in the Examples described herein.
- mice Mus musculus RXR ⁇ (“MmRXR ⁇ -EF”; SEQ ID NO: 9) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ED NO: 51) and placed under the control of an SV40e promoter (SEQ ED NO: 60).
- Five consensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprising SEQ ID NO: 55) were fused to a synthetic Elb minimal promoter (SEQ ID NO: 61) and placed upstream of the luciferase gene (SEQ ID NO: 62).
- CfUSP-EF SEQ ID NO: 66
- VP16AD SEQ BD NO: 51
- SEQ ED NO: 60 An SV40e promoter
- 5XGAL4RE 5 copies of a GAL4RE comprising SEQ ID NO: 55
- M. musculus MmRXR ⁇ (“MmRXR ⁇ - EF", SEQ ID NO: 9).
- CfUSP-EF SEQ ED NO: 66
- VP16AD SEQ ID NO: 51
- SEQ ED NO: 60 An SV40e promoter
- 5XGAL4RE 5 copies of a GAL4RE comprising SEQ ED NO: 55
- MmRXR ⁇ -EF and helices 8 through 12 of LmUSP-EF (SEQ ID NO: 45).
- SV40e promoter (SEQ BD NO: 60).
- Chimeric EF domains comprising helices 1 through 7 of -MmRXR ⁇ - EF and helices 8 through 12 of LmUSP-EF (SEQ ED NO: 45) were fused to the transactivation domain from VP16 ("VP16AD"; SEQ ID NO: 51) and placed under the control of an SV40e promoter (SEQ ED - NO: 60).
- 5XGAL4RE Five consensus GAL4 response element binding sites
- 5XGAL4RE comprising 5 copies of a GAL4RE comprising SEQ ED NO: 55
- SEQ ED NO: 61 a synthetic Elb minimal promoter
- SEQ ID NO: 62 a synthetic Elb minimal promoter
- GAL4CfEcR-CDEF/VP16HsRXR ⁇ -EF The C, D, E, and F domains from spruce budworm Choristoneurafumiferana EcR ("CfEcR-CDEF”; SEQ ID NO: 59) were fused to a GAL4 DNA binding domain ("GaWDNABD” or "GaWDBD”; SEQ ED NO: 47) and placed under the control of an SV40e promoter (SEQ ED NO: 60).
- HsRXR ⁇ -EF human Homo sapiens RXR ⁇
- VP16AD human Homo sapiens VP16
- SEQ ED NO: 60 The EF domains from human Homo sapiens RXR ⁇ (“HsRXR ⁇ -EF”; SEQ ID NO: 13) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ ID NO: 51) and placed under the control of an SV40e promoter (SEQ ED NO: 60).
- Five consensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprising SEQ ID NO: 55) were fused to a synthetic Elb minimal promoter (SEQ ED NO: 61) and placed upstream of the luciferase gene (SEQ ED NO: 62).
- invertebrate RXRs and their non- Lepidopteran and non-Dipteran RXR homologs can function similarly to or better than vertebrate RXRs in an ecdysone receptor-based inducible gene expression modulation system in both yeast and mammalian cells (U.S. provisional application serial No. 60/294,814).
- LmUSP is a better partner for CfEcR than mouse RXR in mammalian cells.
- Applicants have constructed and analyzed vertebrate RXR/invertebrate RXR chimeras (referred to herein interchangeably as “chimeric RXR's" or “RXR chimeras”) in an EcR-based inducible gene expression modulation system. Briefly, gene induction potential (magnitude of induction) and ligand specificity and sensitivity were examined using a non-steroidal ligand in a dose-dependent induction of reporter gene expression in the transfected NIH3T3 cells and A549 cells.
- Ch#l, Ch#2, and Ch#3 in Figures 1-3 were picked and compared with the parental -V--nRXR ⁇ -EF and LmUSP-EF switches (switches 1.1 and 1.2, respectively, as prepared in Example 1).
- the RXR chimera and parent DNAs were transfected into mouse NIH3T3 cells along with Gal4/CfEcR-CDEF and the reporter plasmid pFRLuc.
- the transfected cells were grown in the presence of 0, 0.2, 1, 5, and 10 ⁇ M non-steroidal ligand N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine (GS-ETM ligand).
- the cells were harvested at 48 hours post treatment and the reporter activity was assayed. The numbers on top of bars correspond to the maximum fold activation/induction for that treatment.
- Transfections DNAs corresponding to the various switch constructs outlined in Example 1, specifically switches 1.1 through 1.6 were transfected into mouse NIH3T3 cells (ATCC) and human A549 cells (ATCC) as follows. Cells were harvested when they reached 50% confluency and plated in 6-, 12- or 24- well plates at 125,000, 50,000, or 25,000 cells, respectively, in 2.5, 1.0, or 0.5 ml of growth medium containing 10% fetal bovine serum (FBS), respectively. NIH3T3 cells were grown in Dulbecco's modified Eagle medium (DMEM; LifeTechnologies) and A549 cells were grown in F12K nutrient mixture (LifeTechnologies). The next day, the cells were rinsed with growth medium and transfected for four hours.
- DMEM Dulbecco's modified Eagle medium
- A549 cells were grown in F12K nutrient mixture (LifeTechnologies). The next day, the cells were rinsed with growth medium and transfected for four hours.
- SuperfectTM (Qiagen Inc.) was found to be the best transfection reagent for 3T3 cells and A549 cells. For 12- well plates, 4 ⁇ l of SuperfectTM was mixed with 100 ⁇ l of growth medium. 1.0 ⁇ g of reporter construct and 0.25 ⁇ g of each receptor construct of the receptor pair to be analyzed were added to the transfection mix. A second reporter construct was added [pTKRL (Promega), 0.1 ⁇ g/transfection mix] that comprises a Renilla luciferase gene operably linked and placed under the control of a thymidine kinase (TK) constitutive promoter and was used for normalization. The contents of the transfection mix were mixed in a vortex mixer and let stand at room temperature for 30 min.
- TKRL thymidine kinase
- the transfection mix was added to the cells maintained in 400 ⁇ l growth medium. The cells were maintained at 37°C and 5% C0 2 for four hours.
- DMSO dimethylsulfoxide
- Ligand The non-steroidal ligand N-(2-ethyl-3-methoxybenzoyl)-N' -(3, 5-dimethylbenzoyl)-N'-t- butylhydrazine (GSTM-E non-steroidal ligand) is a synthetic stable ecdysteroid ligand synthesized at
- Reporter Assays Cells were harvested 48 hours after adding ligands. 125, 250, or 500 ⁇ l of passive lysis buffer (part of Dual-luciferaseTM reporter assay system from Promega Co ⁇ oration) were added to each well of 24- or 12- or 6-well plates respectively. The plates were placed on a rotary shaker for 15 minutes. Twenty ⁇ l of lysate were assayed. Luciferase activity was measured using Dual-luciferaseTM reporter assay system from Promega Corporation following the manufacturer's instructions, ⁇ - Galactosidase was measured using Galacto-StarTM assay kit from TROPIX following the manufacturer's instructions.
- a chimeric RXR ligand binding domain may be used in place of a vertebrate RXR or an invertebrate RXR in an EcR-based inducible gene expression modulation system.
- This novel EcR/chimeric RXR-based gene expression system provides an improved system characterized by both increased ligand sensitivity and increased magnitude of induction.
- the EcR/chimeric RXR-based inducible gene expression system of the present invention is an improvement over both the EcR/vertebrate RXR- and EcR/invertebrate RXR-based gene expression modulation systems since less ligand is required for transactivation and increased levels of transactivation can be achieved.
- EcR/chimeric RXR inducible gene expression system of the present invention is useful in applications where modulation of gene expression levels is desired in both yeast and mammalian cells.
- Applicants' invention is also contemplated to work in other cells, including but not limited to bacterial cells, fungal cells, and animal cells.
- constructs were transfected in NIH3T3 cells and transactivation potential was assayed in the presence of 0, 0.2, 1, and 10 ⁇ MN-(2-ethyl-3-methoxybenzoyl)N'-(3,5-dimethylbenzoyl)-N'-tert- butylhydrazine non-steroidal ligand.
- the F-domain chimeras (gene switches 1.4-1.6) were compared to the MmRXR ⁇ -EF(l-7)-LmUSP-EF(8-12) chimeric RXR LBD of gene switch 1.3.
- Plasmid pFRLUC (Stratagene) encoding a luciferase polypeptide was used as a reporter gene construct and pTKRL (Promega) encoding a Renilla luciferase polypeptide under the control of the constitutive TK promoter was used to normalize the transfections as described above.
- the cells were harvested, lysed and luciferase reporter activity was measured in the cell lysates. Total fly luciferase relative light units are presented. The nmnber on the top of each bar is the maximum fold induction for that treatment.
- the analysis was performed in triplicate and mean luciferase counts [total relative light units (RLU)] were determined as described above.
- This Example describes the construction of four EcR-DEF-based gene switches comprising the DEF domains from Choristoneurafumiferana (Lepidoptera), Drosophila melanogaster (Diptera), Tenebrio molitor (Coleoptera), and Amblyomma americanum (Ixodidae) fused to a GAL4 DNA binding domain, i addition, the EF domains of vertebrate RXRs, invertebrate RXRs, or invertebrate USPs from Choristoneurafumiferana USP, Drosophila melanogaster USP, Locusta migratoria USP (OrthopteraJ, Mus musculus RXR ⁇ (Vertebrata), a chimera between Mm-RXR ⁇ and LmUSP (Chimera; of switch 1.13), Amblyomma americanum RXR homolog 1 (Ixodidae), Amblyomma americanum RXR homo
- the receptor combinations were compared for their ability to transactivate the reporter plasmid pFRLuc in mouse NIH3T3 cells in the presence of 0, 0.2, 1, or 10 ⁇ M PonA steroidal ligand (Sigma Chemical Company) or 0, 0.04, 0.2, 1, or 10 ⁇ M N-(2-ethyl-3- methoxybenzoyl)N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine non-steroidal ligand as described above.
- the cells were harvested, lysed and luciferase reporter activity was measured in the cell lysates. Total fly luciferase relative light units are presented. The number on the top of each bar is the maximum fold induction for that treatment.
- the analysis was performed in triplicate and mean luciferase counts [total relative light units (RLU)] were determined as described above.
- FIGS. 6-8 show the results of these analyses.
- the MmRXR-LmUSP chimera was the best partner for CfEcR (11,000 fold induction, Figure 6), DmEcR (1759 fold induction, Figure 7).
- the RXR chimera produced higher background levels in the absence of ligand (see Figure 8).
- the CfEcR/chimeric RXR-based switch (switch 1.13) was more sensitive to non-steroid than PonA whereas, the DmEcR/chimeric RXR-based switch (switch 1.20) was more sensitive to PonA than non-steroid. Since these two switch formats produce decent levels of induction and show differential sensitivity to steroids and non-steroids, these may be exploited for applications in which two or more gene switches are desired.
- the TmEcR/chimeric RXR-based switch (switch 1.21; Figure 8) is more sensitive to PonA and less sensitive to non-steroid and works best when partnered with either MmRXR ⁇ , AmaRXRl, or AmaRXR2.
- the AmaEcR/chimeric RXR-based switch (switch 1.22; Figure 8) is also more sensitive to PonA and less sensitive to non-steroid and works best when partnered with either an LmUSP, MmRXR, AmaRXRl or AmaRXR2-based gene expression cassette.
- TmEcR/ and AmaEcR/chimeric RXR-based gene switches appear to form a group of ecdysone receptors that is different from lepidopteran and dipteran EcR/chimeric RXR-based gene switches group (CfEcR/chimeric RXR and DmEcR/chimeric RXR, respectively).
- EcR/chimeric RXR-based gene switches group CfEcR/chimeric RXR and DmEcR/chimeric RXR, respectively.
- EcR/chimeric RXR-based gene switches are advantageous for use in applications in which two or more gene switches are desired.
- This Example describes Applicants' further analysis of gene expression cassettes encoding various chimeric RXR polypeptides comprising a mouse RXR ⁇ isoform polypeptide fragment or a human RXR ⁇ isoform polypeptide fragment and an LmUSP polypeptide fragment in mouse NIH3T3 cells.
- RXR chimeras were constructed in an effort to identify the helix or helices of the EF domain that account for the observed transactivational differences between vertebrate and invertebrate RXRs.
- five different gene expression cassettes encoding a chimeric RXR ligand binding domain were constructed as described in Example 1.
- the five chimeric RXR ligand binding domains encoded by these gene expression cassettes and the respective vertebrate RXR and invertebrate RXR fragments they comprise are depicted in Table 1.
- the transfected cells were cultured in the presence of either a) 0, 0.2, 1, or 10 ⁇ M non- steroidal ligand (Figure 9), or b) 0, 0.2, 1, or 10 ⁇ M steroid ligand PonA or 0, 0.4, 0.2, 1, or 10 ⁇ M non- steroid ligand ( Figures 10 and 11) for 48 hours.
- the reporter gene activity was measured and total RLU are shown. The number on top of each bar is the maximum fold induction for that treatment and is the mean of three replicates.
- chimera 9 demonstrated maximum induction (see Figure 10).
- Chimeras 6 and 8 also produced good induction and lower background, as a result the fold induction was higher for these two chimeras when compared to chimera 9.
- Chimeras 10 and 11 produced lower levels of reporter activity.
- chimera 8 produced the reporter activity (see Figure 11).
- Chimera 9 also performed well, whereas chimeras 6, 10 and 11 demonstrated lower levels of reporter activity.
- the selection of a particular chimeric RXR ligand binding domain can also influence the performance EcR in response to a particular ligand.
- CfEcR in combination with chimera M responded well to non-steroid but not to PonA (see Figure 10).
- DmEcR in combination with chimera 11 responded well to PonA but not to non-steroid (see Figure 11).
- This Example demonstrates the effect of introduction of a second ligand into the host cell comprising an EcR/chimeric RXR-based inducible gene expression modulation system of the invention.
- Applicants have determined the effect of 9-cis-retinoic acid on the transactivation potential of the GAL4CfEcR-DEF/VP16HsRXR ⁇ -(l-8)-LmUSP-(9-12)-EF ( ⁇ chimera 9; switch 1.27) gene switch along with pFRLuc in NIH 3T3 cells in the presence of non-steroid (GSE) for 48 hours.
- GSE non-steroid
- GAL4CfEcR-DEF, pFRLuc and VP16HsRXR ⁇ -(l-8)-LmUSP-(9-12)-EF were transfected into NIH3T3 cells and the transfected cells were treated with 0, 0.04, 0.2, 1, 5 and 25 ⁇ M non-steroidal ligand (GSE) and 0, 1, 5 and 25 ⁇ M 9-cis-retinoic acid (Sigma Chemical Company).
- GSE non-steroidal ligand
- 9-cis-retinoic acid Sigma Chemical Company
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DK02714990.5T DK1572862T3 (en) | 2001-02-20 | 2002-02-20 | Chimeric, retinoid x receptors and their use in a novel ecdysone receptor-based inducible gene expression system |
JP2002566321A JP4955905B2 (en) | 2001-02-20 | 2002-02-20 | Chimeric retinoid X receptors and their use in novel ecdysone receptor-based inducible gene expression systems |
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ES02714990T ES2392508T3 (en) | 2001-02-20 | 2002-02-20 | Chimeric X retinoid receptors and their use in an inducible gene expression system based on novel ecdysone receptors |
CA2438119A CA2438119C (en) | 2001-02-20 | 2002-02-20 | Chimeric retinoid x receptors and their use in a novel ecdysone receptor-based inducible gene expression system |
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US11/841,597 US8236556B2 (en) | 2001-02-20 | 2007-08-20 | Method of modulating gene expression using an ecdysone receptor-based inducible gene expression system |
US11/841,648 US8598409B2 (en) | 2001-02-20 | 2007-08-24 | Non-human organism comprising a gene expression modulation system encoding a chimeric retinoid X receptor |
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US13/618,742 US20130232588A1 (en) | 2001-02-20 | 2012-09-14 | Chimeric Retinoid X Receptors and Their Use in a Novel Ecdysone Receptor-Based Inducible Gene Expression System |
US14/551,247 US20150150152A1 (en) | 2001-02-20 | 2014-11-24 | Chimeric Retinoid X Receptors and Their Use in a Novel Ecdysone Receptor-Based Inducible Gene Expression System |
US15/293,602 US20170096673A1 (en) | 2001-02-20 | 2016-10-14 | Chimeric Retinoid X Receptors and Their Use in a Novel Ecdysone Receptor-Based Inducible Gene Expression System |
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