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WO2000049161A1 - Rapporteurs de synthese destines a controler les niveaux de la camp - Google Patents

Rapporteurs de synthese destines a controler les niveaux de la camp Download PDF

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Publication number
WO2000049161A1
WO2000049161A1 PCT/US2000/004165 US0004165W WO0049161A1 WO 2000049161 A1 WO2000049161 A1 WO 2000049161A1 US 0004165 W US0004165 W US 0004165W WO 0049161 A1 WO0049161 A1 WO 0049161A1
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Prior art keywords
cell line
cassette
camp
stimulus
group
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English (en)
Inventor
Xianqiang Li
Xiaoning Zhao
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Takara Bio USA Inc
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Clontech Laboratories Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • 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.
  • 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:
  • 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.
  • 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.
  • cAMP serves as a second messenger, produced as a result of th e activation of adenylate cyclase by a 7-span cell-surface receptor.
  • cAMP activates a cyclic AMP-dependent protein kinase that phosphorylates, and thus activates or inactivates, key enzymes of hormone-regulated metabolic pathways.
  • the level of cAMP is regulated by nutrient supply and controls gene expression through interaction with th e cyclic AMP activator protein (CAP).
  • CAP th e cyclic AMP activator protein
  • 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 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).
  • Em extinction coefficient
  • 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 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.
  • MODC mouse ornithine decarboxylase
  • 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.
  • 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.
  • CRE cAMP response element
  • 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.
  • 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.
  • CRE cAMP response elements
  • a reporter cassette for monitoring cAMP levels comprising, in operable linkage: a) five cAMP response elements (CREs); b) an gonadotropin ⁇ -gene promoter; and c) a gene encoding d2EGFP.
  • CREs cAMP response elements
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • CRE cAMP response element
  • 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;
  • the promoter is the gonadotropin ⁇ -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 ⁇ -gene promoter; and c) a gene encoding d2EGFP.
  • CREs cAMP response elements
  • 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.
  • 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.
  • 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.
  • w h en the cassette-containing medium is in vivo, combining is b y transformation, transfection, electroporation and transduction.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 a t levels detectable above background.
  • 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.
  • primer 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.
  • 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.
  • the transforming DNA may be maintained on an episomal element such as a vector or plasmid.
  • 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.
  • 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".
  • 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.
  • 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.
  • th e gene 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.
  • 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 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.
  • CRE cAMP response element
  • ⁇ CRE5-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 ⁇ -gene promoter (also called thyroid-stimulating hormone (TSH)- ⁇ subunit).
  • TSH thyroid-stimulating hormone
  • 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.
  • 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.

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Abstract

La présente invention concerne un produit de synthèse qui permet de contrôler le niveau de la cAMP (adénosine 3',5'-monophosphate cyclique) in vivo. L'élévation de la cAMP dans les cellules entraîne la phosphorylation du facteur de transcription liant l'élément de réponse de la cAMP (CREB). Le facteur CREB phosphorylé se lie alors aux sites d'élément de réponse de la cAMP (CRE) du rapporteur de synthèse, ce qui provoque ensuite la transcription et la traduction du gène rapporteur d2EGFP. Le rapporteur de synthèse de l'invention peut être utilisé dans des analyses à haut rendement permettant de cribler des facteurs et des mutants impliqués dans la voie de transduction de signal de la cAMP.
PCT/US2000/004165 1999-02-17 2000-02-17 Rapporteurs de synthese destines a controler les niveaux de la camp Ceased WO2000049161A1 (fr)

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US12046399P 1999-02-17 1999-02-17
US60/120,463 1999-02-17

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WO2000049161A1 true WO2000049161A1 (fr) 2000-08-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113490746A (zh) * 2018-12-19 2021-10-08 植物心语公司 敏感植物和用于基于敏感植物的特征来识别作物中的应激原的方法
US12360044B2 (en) 2018-12-19 2025-07-15 InnerPlant, Inc. Method for identifying stressors in an agricultural environment based on characteristics of microbe sensors

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5728545A (en) * 1993-06-18 1998-03-17 The Salk Institute Of Biological Studies Cloning and recombinant production of CRF receptor (S)
US5989814A (en) * 1997-04-01 1999-11-23 Reagents Of The University Of California Screening methods in eucaryotic cells
US6025157A (en) * 1997-02-18 2000-02-15 Genentech, Inc. Neurturin receptor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5728545A (en) * 1993-06-18 1998-03-17 The Salk Institute Of Biological Studies Cloning and recombinant production of CRF receptor (S)
US6025157A (en) * 1997-02-18 2000-02-15 Genentech, Inc. Neurturin receptor
US5989814A (en) * 1997-04-01 1999-11-23 Reagents Of The University Of California Screening methods in eucaryotic cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GALIEN, R. ET. AL.: "Differential effects of c-jun and CREB on c-AMP response element by Ha-Pas.", ONCOGENE, vol. 9, 1994, pages 1102 - 1108, XP002928205 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113490746A (zh) * 2018-12-19 2021-10-08 植物心语公司 敏感植物和用于基于敏感植物的特征来识别作物中的应激原的方法
US12360044B2 (en) 2018-12-19 2025-07-15 InnerPlant, Inc. Method for identifying stressors in an agricultural environment based on characteristics of microbe sensors

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