WO2000049161A1

WO2000049161A1 - REPORTER CONSTRUCTS TO MONITOR cAMP LEVELS

Info

Publication number: WO2000049161A1
Application number: PCT/US2000/004165
Authority: WO
Inventors: Xianqiang Li; Xiaoning Zhao
Original assignee: Clontech Laboratories Inc
Current assignee: Takara Bio USA Inc
Priority date: 1999-02-17
Filing date: 2000-02-17
Publication date: 2000-08-24
Anticipated expiration: 2001-08-17

Abstract

The construct of the present invention allows for the level of cAMP to be monitored in vivo. cAMP elevation within cells results in the phosphorylation of the cAMP response element binding (CREB) transcription factor. The phosphorylated CREB factor then binds the cAMP response element (CRE) sites of the reporter construct, which subsequently induces the transcription and translation of the d2EGFP reporter gene. This reporter construct can be used in high-throughput assays to screen for factors and mutants involved in the cAMP signal transduction pathway.

Description

REPORTER CONSTRUCTS TO MONITOR cAMP LEVELS

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to DNA reporter contructs and the use of such reporter constructs to monitor cAMP levels in vitro or in vivo.

Description of the Related Art

Cells are sensitive to an extraordinary number of chemical signals; these signals control cell and tissue behavior and integrate body function. These chemical signals may originate close to the responding cells (e.g. neurotransmitters , prostaglandins), be distributed throughout multicellular bodies (e.g. classical hormones), or travel between organisms (e.g. odours). The 'target cells' for each signal are the cells that be ar receptors for the signal agent and each extracellular stimulus acts at a unique receptor or type of receptor. Many cell-surface receptors signal through relay systems in which three components act in sequence:

'receptor→ G_* protein→ effector' . G_* proteins are guanine-nucleotide dependent coupling proteins , where the subscript identifies a particular G protein. The effector "protein" is usually an enzyme or an ion channel. The hundreds or thousands of receptors that function in this way seem all to b e members of a single structural family of polypeptides, typified b y the rhodopsins. These '7-span' receptors are embedded within th e plasma membrane, with their polypeptide chain traversing th e membrane seven times. The sites through which they respond to extracellular stimuli are exposed to the external medium, an d their coupling to G proteins, and thence to effectors, is achieved primarily through the intracellular polypeptide loop that links th e fifth and sixth transmembrane domains.

In the mid- 1950s, Sutherland' s laboratory discovered that catecholamines, acting through β-adrenergic receptors , transmit information to the cell interior by stimulating th e synthesis of adenosine 3',5'-cyclic monophosphate (cyclic AMP, cAMP). Adenosine 3', 5'-cyclic monophosphate is a freely diffusing, water-soluble nucleotide formed from ATP by the action of adenylate cyclase, and is converted to AMP by cAMP phosphodiesterase.

In eukaryotic cells (except, it seems, in higher plants), cAMP serves as a second messenger, produced as a result of th e activation of adenylate cyclase by a 7-span cell-surface receptor. At micromolar concentrations, cAMP activates a cyclic AMP- dependent protein kinase that phosphorylates, and thus activates or inactivates, key enzymes of hormone-regulated metabolic pathways. In bacteria, the level of cAMP is regulated by nutrient supply and controls gene expression through interaction with th e cyclic AMP activator protein (CAP).

A substantial number of the many 7-span receptor species at the cell surface are now known to control cells b y influencing adenylate cyclase activity; some 7-span receptor species stimulate cAMP formation and others inhibit its formation. Thus, most or all of the extracellular information delivered to these diverse receptors is summarized for the cell interior simply as a rise or fall in the intracellular concentation of cAMP. Therefore, monitoring cAMP in cells can provide valuable information about one or more of the steps involved in the cell signalling process, including screening for signal molecules th at induce or inhibit intracellular levels of cAMP, and detecting mutants in receptors, G proteins or intracellular enzymes involved in the signal transduction.

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time (1-4). GFP fluoresces bright green upon mere exposure to UN o r blue light-unlike other bioluminescent reporters which require additional proteins, substrates, or cofactors to emit light. GFP fluorescence is stable, species independent and can be monitored noninvasively in living cells. GFP fluorescence persists in formaldehyde-fixed cells and is well suited for double-labeling experiments with other fluorescent markers, including the GFP variant enhanced green fluorescent protein (EGFP, GFP_mutl) (5-6).

EGFP encodes a protein which has a single, red-shifted excitation peak and fluoresces about 35 times more intensively than wild type GFP when excited at 488 nm (7), due to an increase in its extinction coefficient (Em). To ensure maximal mammalian expression, the coding region of EGFP contains more than 1 90 silent base mutations which correspond to human codon-usage preferences (7). The red-shifted spectrum and increased expression of EGFP make it ideal for fluorescence microscopy an d fluorescence-activated cell sorting (FACS) (5, 8).

Destabilized EGFP (d2EGFP) contains the PEST domain from mouse ornithine decarboxylase (MODC), fused to the C- terminus of EGFP (9). This domain targets EGFP for rapid turnover effectively reducing EGFP's half-life to two hours. The introduction of d2EGFP greatly increases the utility of GFP i n studying dynamic cellular-events in vivo.

The prior art is deficient in a reporter construct which readily and non-destructively allows monitoring of intracellular levels of cAMP. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The construct described herein was designed to monitor the cAMP level changes in vivo . cAMP elevation within cells results in the phosphorylation of the cAMP response element binding (CREB) transcription factor. The phosphorylated CREB factor then binds the cAMP response element (CRE) sites of th e reporter construct, which subsequently induces the transcription and translation of the reporter gene. This reporter construct can be used in high-throughput assays to screen for factors and mutants involved in the cAMP signal transduction pathway, or to monitor, in vivo, cAMP levels in real time.

One object of the present invention is to provide a reporter construct to monitor the intracellular levels of cAMP.

In an embodiment of the present invention, there is provided a reporter cassette for monitoring cAMP levels, comprising, in operable linkage: a) four, five or six cAMP response elements (CRE); b) a promoter; and c) a gene encoding a fluorescent reporter molecule.

In one embodiment of the present invention, there is provided a reporter cassette for monitoring cAMP levels, comprising, in operable linkage: a) five cAMP response elements (CREs); b) an gonadotropin α-gene promoter; and c) a gene encoding d2EGFP. Other embodiments of the present invention include vectors and cell lines containing the above-described cassettes, host cells containing the vectors, and kits comprising th e cassettes, the vectors, the host cells and/or the cell lines.

In another embodiment of the present invention, th ere is provided a method of monitoring cAMP levels in a medium in response to a stimulus, comprising the steps of: a) combining a reporter cassette, comprising, in operable linkage: 1) four, five o r six cAMP response elements (CRE); 2) a promoter; and 3) a gene encoding a fluorescent reporter molecule with an appropriate medium, thereby producing cassette-containing medium; b ) measuring flourescence of the cassette-containing medium; c) contacting the cassette-containing medium with a stimulus; and d ) measuring fluorescence of the medium. In this embodiment, a greater amount of fluorescence following contact with the stimulus than prior to contact with the stimulus indicates an induction of cAMP levels in response to the stimulus, while less fluorescence following contact with the stimulus than prior to contact with th e stimulus indicates an inhibition of cAMP levels in response to th e stimulus .

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figure 1 shows a schematic of the plasmid CRE5- dEGFP. Figure 2 shows a time course (A) and a dose response (B) of CRE5-dE clone #3 in response to Forskolin.

DETAILED DESCRIPTION OF THE INVENTION

The construct described herein was designed to monitor the cAMP level changes in vivo . cAMP elevation within cells results in the phosphorylation of the cAMP response element binding (CREB) transcription factor. The phosphorylated CREB factor then binds the cAMP response element (CRE) sites of th e reporter construct, which subsequently induces the transcription and translation of the d2EGFP reporter gene. The destabilized form of EGFP eliminates the accumulation of EGFP in cells an d more accurately detects changes in the intracellular level of cAMP. This reporter construct can be used in high-throughput assays to screen for factors and mutants involved in the cAMP signal transduction pathway, or to monitor, in vivo, cAMP levels in real time. The present invention is directed towards a reporter cassette for monitoring cAMP levels, comprising, in operable linkage: a) four, five or six tandem cAMP response elements (CREs) b) a promoter; and c) a gene encoding a destabilized fluorescent reporter molecule;. Preferably, the promoter is the gonadotropin α-gene promoter, the thymidine kinase promoter or TATAA, and the gene encoding a fluorescent reporter molecule is d2EGFP o r dlEGFP. Representative means of detecting fluorescence include spectroscopy, fluorometry, plate reading and flow cytometry. The present invention is further directed towards a reporter cassette for monitoring cAMP levels, comprising, in operable linkage: a) five cAMP response elements (CREs); b) a n gonadotropin α-gene promoter; and c) a gene encoding d2EGFP. The present invention is also directed towards vectors and cell lines comprising the above-described cassettes, host cells comprising the vectors, and kits comprising the cassettes, th e vectors, the host cells and/or the cell lines.

The present invention is also directed to a method of monitoring cAMP levels in a medium in response to a stimulus, comprising the steps of: a) combining a reporter cassette, comprising, in operable linkage: 1) four, five or six cAMP response elements (CRE); 2) a promoter; and 3) a gene encoding a fluorescent reporter molecule, with an appropriate medium, thereby producing cassette-containing medium; b) measuring flourescence of the cassette-containing medium; c) contacting th e cassette-containing medium with a stimulus; and d) measuring fluorescence of the medium. Generally, a greater amount of fluorescence following contact with the stimulus than prior to contact with the stimulus indicates an induction of cAMP levels i n response to the stimulus, while less fluorescence following contact with the stimulus than prior to contact with the stimulus indicates an inhibition of cAMP levels in response to the stimulus . Generally, the medium may be combined with the cassette in vitro and in vivo. Representative stimuli include pharmaceutical drugs, chemicals, known inducers of cAMP or cAMP pathways and known inhibitors of cAMP or cAMP pathways. Representative means of detecting fluorescence include spectroscopy, fluorometry, plate reading and flow cytometry. Generally, w h en the cassette-containing medium is in vivo, combining is b y transformation, transfection, electroporation and transduction.

As used herein, "reporter" refers to a molecule (usually a protein) that is expressed in response to or as a result of a particular biological or molecular event.

As used herein, the term "cassette" refers to a combination of different DNA elements to produce a funtional genetic unit, typically the cassette is flanked by restriction sites for cloning purposes.

In accordance with the present invention, there m ay be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & SJ. Higgins eds. (1985)] ; "Transcription and Translation" [B.D. Hames & SJ. Higgins eds. (1984)] ; "Animal Cell Culture" [R.I. Freshney, ed . (1986)] ; "Immobilized Cells And Enzymes" [IRL Press, ( 1986)] ; B. Perbal, "A Practical Guide To Molecular Cloning" ( 1984) . Therefore, if appearing herein, the following terms shall have th e definitions set out below.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) i n its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of th e molecule, and does not limit it to any particular tertiary forms . Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

A "vector" is a replicon, such as plasmid, phage o r cosmid, 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, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. An "origin of replication" refers to those DNA sequences that participate in DNA synthesis. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "operably linked" and "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription an d translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection i n transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth. A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A "cDNA" is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript. An "exon" is an expressed sequence transcribed from the gene locus, whereas an "intron" is a non-expressed sequence that is from th e gene locus.

Transcriptional and translational control sequences ar e DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A "cis-element" is a nucleotide sequence, also termed a "consensus sequence" o r "motif", that interacts with other proteins which can upregulate o r downregulate expression of a specicif gene locus. A " signal sequence" can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes an d eukaryotes . 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. For purposes of defining the present invention, 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 a t levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well a s protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, b u t not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the - 1 0 and -35 consensus sequences. The term "oligonucleotide" is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of th e oligonucleotide. The term "primer" as used herein refers to a n oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in th e presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must b e sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature , source of primer and use the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

A cell has been "transformed" or "transfected" with exogenous or heterologous DNA when such DNA has b een introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. I n prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA h as become integrated into a chromosome so that it is inherited b y daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed "transgenic".

As used herein, the term "host" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant an d animal cells. A recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Prokaryotic hosts may include E coli, S. tymph imurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells, and more preferentially, plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.

Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, 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 th e 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., Maniatis et al., supra; DNA Cloning, Nols. I & II, supra; Nucleic Acid Hybridization, supra. A "heterologous' region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, th e gene will usually be flanked by DNA that does not flank th e mammalian genomic DNA in the genome of the source organism. In another example, the coding sequence is a construct where th e coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion: EXAMPLE 1

Description of the Reporter Construct

The construct of the present invention was designed to monitor the cAMP level changes in vivo. cAMP elevation within cells results in the phosphorylation of the cAMP response element binding (CREB) transcription factor. The phosphorylated CREB factor then binds the cAMP response element (CRE) sites of th e reporter construct, which subsequently induces the transcription and translation of the d2EGFP reporter gene. The destabilized form of EGFP eliminates the accumulation of EGFP in cells and more accurately detects changes in the intracellular level of cAMP.

EXAMPLE 2

Construction of the Reporter Construct

ρCRE5-dEGFP was constructed using the pTK-SEAP plasmid. The Bglll-Hindlll fragment of pTK-SEAP containing th e TK promoter was replaced with a BamRl-HindUl fragment containing five copies of the cAMP response elements (CREs) followed by the gonadotropin α-gene promoter (also called thyroid-stimulating hormone (TSH)-α subunit). The d2EGFP reporter gene was then cloned directly adjacent the gonadotropin α-gene promoter, thereby replacing SEAP. EXAMPLE 3

Generation of a Cell Line Expressing pCRE5-dEGFP

A stable cell line was generated by transfecting pCRE5- dEGFP, together with pSV2-neo, into CHO-Kl cells. Transfected cells were selected under G418 at 500 mg/ml concentration and individual clones were isolated and analyzed for Forskolin induction. Three cell clones were selected which showed a time course of induction, as well as a dose response of induction, b y Forskolin. Clone CRE5-dE #3 has the highest fluorescence intensity and a 5-fold increase in fluorescence is observed after cAMP induction by Forskolin (Figure 2). The highest fold inducibility (10-fold) and the lowest amount of background EGFP expression was observed with clone CRE5-dE #38. However, following induction, the intensity of fluorescence is lower than that of clone #3. Clone #38-2 is an intermediate of clones #3 and #38, and h as an 8-fold induction of fluorescence. After induction, th e fluorescence intensity of clone #38-2 is at a similar level to clone #3.

EXAMPLE 4

Sequence of the pCRE5-dEGFP plasmid

GGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCCTCGAGCCCATGGCCGTCA TACTGTGACGTCCCCATGGCCGTCATACTGTGACGTCCCCATGGCCGTCATACTGTGAC GTCCCCATGGTCGTCATACTGTGACGTCCCCATGGCCGTCATACTGTGACGTCCCGCGG TCGACTACCCTTCAATCATTGGATGGAATTTCCTGTTGATCCCAGGGCTTAGATGCAGG TGGAAACACTCTGCTGGTATAAAAGCAGGTGAGGACTTCATTAACTGCAGTTACTGAGA ACTCATAAGACGAAGCTAAAATCCCTCTTCGGAAGCTTCCCCGAATTCCGGATGGTGAG CAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACG TAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAG CTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGT GACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGC ACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTC AAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACA AGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGC CGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC ACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAA GAAGCTTAGCCATGGCTTCCCGCCGGAGGTGGAGGAGCAGGATGATGGCACGCTGCCCA TGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGG ATCAATGTGTAGTCTAGAGTCGGGGCGGCCGGCCGCTTCGAGCAGACATGATAAGATAC ATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGA AATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACA ACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAA AGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGTCGACCGATGCCCT TGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCC GCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTT CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA GCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG

TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGT GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATC CGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG

CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAA GCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA GAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA TGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGC CTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAG CCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTC TATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACG TTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGC GGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCAC TCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAG TTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAG TGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTG AGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTT CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATT TATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACA AATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCG CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCC CCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAG ACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCA AACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTT AACAAAATATTAACGTTTACAATTTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA

AGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCCCAAGCTACCATGATAAGTAA GTAATATTAAGGTACGGGAGGTACTTGGAGCGGCCGCAATAAAATATCTTTATTTTCAT TACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGTACTAACATACGCTCTCCATCAA AACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGC CAGAACATTTCTCTATCGATA

The following references were cited herein:

1 . Chalfie, et al., (1994) Science 263: 802-805.

2. Prasher, et al., (1992) Gene 111 : 229-233. 3. Inouye, et al., (1994) FEBS Letters 341 : 277-280. 4. Wang, et al., (1994) Nature 369: 400-403.

5. Yang, et al., (1996) Nucleic Acid Res. 24(22): 4592-4593.

6. Cormack, et al., (1996) Gene 173: 33-38.

7. Hass, et al., (1996) Curr. Biol. 6: 315-324. 8. Galbraith, et al., (1995) Methods. Cell Biol. 50: 1-12. 9. Living Colors Destabilized EGFP Vectors (April 1998), CLONTECHniques XIII(2): 16-17.

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the s ame extent as if each individual publication was specifically an d individually indicated to be incorporated by reference.

One skilled in the art will appreciate readily that th e present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The pre sent examples, along with the methods, procedures, treatments , molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined b y the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1 . A reporter cassette for monitoring cAMP levels, comprising, in operable linkage:

a) cAMP response elements (CREs), wherein th e number of CREs is selected from the group consisting of four, five and six; b ) a promoter; and

c) a gene encoding a fluorescent reporter molecule.

2. The cassette of claim 1, wherein said promoter is selected from the group consisting of the gonadotropin α-gene promoter, the thymidine kinase promoter and TATAA.

3. The cassette of claim 1, wherein fluorescence is detected by means selected from the group consisting of spectroscopy, fluorometry, plate reading and flow cytometry.

4. The cassette of claim 1 , wherein said gene encoding a fluorescent reporter molecule is selected from th e group consisting of d2EGFP and dlEGFP.

5. A vector comprising the cassette of claim 1.

A host cell comprising the vector of claim 5.

7. A cell line comprising the cassette of claim 1.

8. The cell line of claim 7, wherein said cell line is selected from the group consisting of a prokaryotic cell line and a eukaryotic cell line.

9. The cell line of claim 7, wherein said cell line is CHO-Kl.

10. A kit comprising the cassette of claim 1.

1 1 . A kit comprising the vector of claim 5.

12. A kit comprising the host cell of claim 6.

13. A kit comprising the cell line of claim 7.

14. A kit comprising the cell line of claim 9.

15. A reporter cassette for monitoring cAMP levels, comprising, in operable linkage:

a) five cAMP response elements (CREs);

b ) a gonadotropin α-gene promoter; and

c) a gene encoding d2EGFP.

16. The cassette of claim 15, wherein fluorescence is detected by means selected from the group consisting of spectroscopy, fluorometry, plate reading and flow cytometry.

17. A vector comprising the cassette of claim 15.

1 8. A host cell comprising the vector of claim 17.

19. A cell line comprising the cassette of claim 15.

20. The cell line of claim 19, wherein said cell line is selected from the group consisting of a prokaryotic cell line and a eukaryotic cell line.

21 . The cell line of claim 19, wherein said cell line is CHO-Kl.

2 2 A kit comprising the cassette of claim 15.

23. A kit comprising the vector of claim 17.

24. A kit comprising the host cell of claim 18.

25. A kit comprising the cell line of claim 19.

26. A kit comprising the cell line of claim 21.

27. A method of monitoring cAMP levels in a medium in response to a stimulus, comprising the steps of:

a) combining the cassette of claim 1 with a medium;

b ) measuring flourescence of said medium;

c) contacting said medium with a stimulus; and

d) measuring fluorescence of said medium, wherein a greater amount of fluorescence following contact with said stimulus than prior to contact with said stimulus indicates a n induction of cAMP levels in response to said stimulus, whereas less fluorescence following contact with said stimulus than prior to contact with said stimulus indicates an inhibition of cAMP levels in response to said stimulus.

28. The method of claim 27, wherein said cassette- containing medium is selected from the group consisting of in vitro and in vivo.

29. The method of claim 27, wherein said stimulus is selected from the group consisting of pharmaceutical drugs, chemicals, known inducers of cAMP or cAMP pathways and known inhibitors of cAMP or cAMP pathways.

30. The method of claim 27, wherein fluorescence is detected by means selected from the group consisting of spectroscopy, fluorometry, plate reading and flow cytometry.

3 1 . The method of claim 28, wherein when said cassette-containing medium is in vivo, said combining is by means selected from the group consisting of transformation, transfection, electroporation and transduction.