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WO1999033973A2 - Cellules clonees produisant un recepteur d'octopamine - Google Patents

Cellules clonees produisant un recepteur d'octopamine Download PDF

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Publication number
WO1999033973A2
WO1999033973A2 PCT/EP1998/008411 EP9808411W WO9933973A2 WO 1999033973 A2 WO1999033973 A2 WO 1999033973A2 EP 9808411 W EP9808411 W EP 9808411W WO 9933973 A2 WO9933973 A2 WO 9933973A2
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gene
octopamine receptor
octopamine
cells
receptor
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PCT/EP1998/008411
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German (de)
English (en)
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WO1999033973A3 (fr
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Arnd Baumann
Stephan Frings
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Forschungszentrum Jülich GmbH
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Publication of WO1999033973A2 publication Critical patent/WO1999033973A2/fr
Publication of WO1999033973A3 publication Critical patent/WO1999033973A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the invention relates to a method for producing octopamine receptor-producing cells according to claims 1 to 10, to octopamine receptor genes according to claims 11 to 14, octopamine receptors according to claims 15 and 16, gene constructs according to claims 17 and 18, transformed cells Claims 19 to 23 and a method for examining the properties of neurotransmitter receptors according to Claims 24 to 27.
  • Nerve cells or neurons form the functional units of the brain.
  • the main task of a neuron is to transfer information to other neurons or to muscle and gland cells.
  • each neuron not only has a diverse repertoire of highly specialized, membrane-bound protein molecules (proteins), but also an extraordinary cellular structure.
  • the following cell areas are morphologically differentiated: dendrite, cell body, axon and synaptic end button.
  • the information is recorded in the dendritic area of the neuron and is passed on as an electrical signal. If a certain threshold value of electrical excitation is exceeded, an action potential is triggered just below the cell body - in the area of the axon hillock.
  • the action potential is characterized by the selective inflow of sodium ions through sodium channels into the cell interior.
  • the sodium ion inflow is affected by a time-delayed potassium ion Outflow through potassium channels counter (repolarization), so that the resting potential is set again.
  • the action potential propagates along the axon down to the synaptic end button. This is the area of the neuron where the cell contacts target cells. The contact point is also known as a synapse.
  • the electrical signal (action potential) cannot be passed on via the synaptic gap. To transmit signals from the neuron to the target cell, the electrical signal must therefore be translated into a chemical signal.
  • the action potential causes the release of messenger substances, the so-called neurotransmitters, from the synaptic end button.
  • the neurotransmitters diffuse to the surface of the target cell and bind there to high-affinity binding proteins, the so-called neurotransmitter receptors, which are specific to the respective neurotransmitter.
  • the first group combines those receptors that can themselves form ion channels (ionotropic receptors). These include the acetylcholine and glutamate receptors, which, when activated, depolarize the target cell and trigger a new action potential. Inhibitory receptors, such as glycine and ⁇ -aminobutyric acid (GABA) -
  • GABA ⁇ -aminobutyric acid
  • Receptors hyperpolarize the target cell and therefore counteract the triggering of a new action potential.
  • the receptors are therefore also called metabotropic receptors.
  • Members of this receptor family have one thing in common Structural characteristic of seven hydrophobic sections in the primary structure (amino acid sequence) that span the cell membrane The change in the intracellular messenger concentration is achieved by the receptors interacting with GTP-binding (G) proteins.
  • adenylate cyclase leads to an increase in the intracellular concentration of cyclic adenosine 3 ', 5'-monophosphate (cAMP).
  • cAMP cyclic adenosine 3 ', 5'-monophosphate
  • Another way is to activate a phospholip ase C and the resulting synthesis of inositol-l, 4,5-triphosphate (IP 3 ) and diacylglycerol (DAG).
  • the metabotropic receptors thus intervene in the regulation of cellular activity and can decisively influence the plasticity of neuronal circuits.
  • modulation of synaptic plasticity is believed to play a crucial role in connection with learning performance and in memory formation.
  • Many of the neurotransmitter / receptor systems known today have been preserved in the course of evolution. However, some only occur in certain animal species.
  • An example of the differentiation of neurotransmitters can be found in the group of biogenic amines.
  • the group of biogenic amines includes the neurotransmitters adrenaline, dopamine, histamine, noradrenaline, octopamine, serotonin and tyramine.
  • octopamine acts as a neurotransmitter, neurohormone and also as a neuromodulator and controls or modulates many biochemical reactions, motor skills and behavior. For example, it is described that octopamine and others. regulates metabolism and energy balance. In addition, octopamine modulates the animals' biorhythms and is crucial for learning and memory building. Only low octopamine concentrations are found in vertebrates. However, it is unclear whether the substance has neuroactive activity.
  • Octopamine receptors have also only been written in evertebrates. These proteins belong to the large gene family of the G protein-coupled receptors. Based on pharmacological binding studies and the investigation of the activated intracellular messenger pathways, two classes of octopamine receptors are distinguished (Evans, PD and Robb, S .: Octopamine receptor subtypes and their modes of action; Neurochemical Research 18, 1993, 869-874).
  • One receptor class - octopamine 1 (OCTl) receptors - causes an increase in the intracellular Ca 2+ concentration - presumably induced via the intracellular messenger IP 3 .
  • the second class of receptors - octopamine 2 (OCT2) receptors - stimulates adenylate cyclase and there is an increase in the intracellular cAMP concentration.
  • the physiologically effective concentration of octopamine is in the range from 10 " 8 to 10" 10 M.
  • octopamine is only detected as a neurotransmitter in invertebrates, it would be of great interest to develop specific pharmaceuticals (ligands) that only bind to octopamine receptors. Since these receptors are not found in vertebrates, such ligands could be used as insecticides without fear of side effects in vertebrates.
  • the homogenate consists of a mixture of different receptors or subtypes, i.e. the receptor populations are present side by side or mixed (Roeder, T. and Nathanson, JA: Characterization of insect neuronal octopamine receptors; Neurochemical Research 18, 1992 , 921-925; Hiripi, L. et al .: Characterization of ty ramine and octopamine receptors in the insect (Locusta migratoria migratorioides) brain; Brain Research 633, 1994, 119-126).
  • tissue homogenates are relatively time-consuming and labor-intensive and sometimes requires the use of protein chemical cleaning and separation processes.
  • a similar problem also applies to the quantification of the intracellular messengers cAMP and IP 3 .
  • the determination of the corresponding concentrations is labor-intensive, time-consuming and often error-prone because different messenger pathways can be activated simultaneously in the tissue preparations.
  • the characterization of the receptors has so far mainly been carried out using the method of radioligand binding.
  • tissue homogenates are incubated with different, radioactively labeled compounds and the specifically bound radioactivity is measured after several filtration and washing steps.
  • the classification of the receptors is only possible if a variety of different substances have been tested to determine whether and which substance is suitable to displace the radioactively labeled compound from the receptor protein (competition experiments). This procedure is also labor-intensive and, due to the use of the radio-labeled compounds, is not harmless for environmental and health reasons (radiation exposure).
  • the use of a detection method that does not require the use of radioactive material is therefore desirable and advantageous.
  • octopamine receptor-producing cells in which at least one gene coding for an octopamine receptor is transformed into a host cell.
  • octopamine receptor genes are preferably cloned from the fruit fly Drosophila melanogaster. After cloning or isolation, in particular genes with the SEQ ID no. 1 and / or 3 specified nucleotide sequences or with one for which under SEQ ID No. 2 and / or 4 indicated amino acid sequence and / or their allele variations coding nucleotide sequence available. These genes can be used primarily for the production of octopamine receptor type 1 and / or 2 (OCT1 or 2) producing cells.
  • a DNA probe for example from a cDNA clone that codes for a Drosophila dopamine receptor (Gotzes, F. et al .: Primary structure and functional characterization of a Drosophila dopamine receptor with high homology to human Dl / 5 receptors; Receptors & Channels 2, 1994, 131-141), can be genomic and cDNA libraries under degraded
  • the constructs can then in particular in a cell line, preferably in the human kidney cell line HEK293, with the calcium phosphate method (Chen, C. and Okayama, H .: High-efficiency transformation of mammalian cells by plasmid DNA; Molecular and Cellular Biology 7 , 1987, 2745-2752).
  • Cell clones that have taken up the DNA stably can be selected by adding the antibiotic Geneticin (G418 sulfate).
  • the DNA molecules that code for the above-mentioned splice variants can also be integrated into the genome of the host cell independently of one another.
  • transient transfection In addition to using the cell line mentioned (HEK 293), it is also conceivable to introduce the DNA which codes for a receptor into other cells, such as, for example, COS, NIH 3T3 or Drosophila S2 cells. This can be done in a stable form, as described above, or alternatively by transient transfection.
  • the disadvantage of transient transfection is that the receptors only last for a short time, i.e. can be examined up to approx. 48 h after the transfection. After that, the progressive cell division means that the expression constructs are only present in the cells in small numbers of copies. This results in a lower expression rate or synthesis rate of the receptor proteins.
  • Another way of expressing the receptor proteins is, for example, using the baculovirus system.
  • the DNA only has to be cloned into an appropriate expression plasmid.
  • the cloning of octopamine receptor genes and their stable expression makes it possible for the first time to examine individual, structurally precisely defined neurotransmitter receptor subtypes. Since the cells according to the invention produce approximately the same amounts of receptors, a further disadvantage of using tissue homogenates is overcome, namely the fluctuation in the receptor density, which can be extremely variable from experiment to experiment. In addition, in particular stable cell lines are grown under constant and easy to control conditions with relatively little effort. This offers the possibility of automating the multiplication process: for example, the cells can be grown in fermenters.
  • Such cells transformed with neurotransmitter receptor genes can then be used in a simple method for examining the properties of neurotransmitter receptors, in which a ligand to be examined is allowed to act on these cells and the effect is determined by determining intracellular messenger substances.
  • a ligand to be examined is allowed to act on these cells and the effect is determined by determining intracellular messenger substances.
  • cloned octopamine receptors activate a cell-specific G protein and subsequently a phospholipase.
  • This enzyme cleaves a membrane-based substrate into two intracellular messenger substances: IP3 and DAG.
  • IP3 binds to an intracellular receptor, which enables the passage of Ca 2+ ions from the cell compartments into the cytoplasm of the cell.
  • the increase in the cytoplasmic Ca 2+ ion concentration can be registered with the aid of a Ca 2+ sensitive indicator, the fluorescent dye FURA-2.
  • the intracellular Ca 2+ ion concentration is well calibrated and the change in concentration quantifiable.
  • the relative change in the Ca 2+ ion concentration is therefore a measure of whether the ligand tested activates the receptor, ie acts as an agonist, or alternatively in the presence of octopamine, which inhibits receptor activity, ie acts as an antagonist. This procedure enables the investigation of a large number of substances (screening) with high throughput numbers.
  • the method according to the invention can also be transferred to other receptors, for example those which bring about the production of the messenger substance cAMP.
  • the increase in the intracellular cAMP concentration can be registered by ion channels that open depending on the cAMP and allow the influx of Ca 2+ ions.
  • the cyclic nucleotide-controlled ion channels have these properties.
  • the increase in cAMP concentrations thus also causes an increase in the intracellular Ca 2+ concentration, which can be measured using the Ca 2+ imaging method.
  • the Ca 2+ flows in this second variant from the extracellular area into the cell interior.
  • the method according to the invention can therefore be applied, inter alia, to all other receptor systems which bring about an increase in the intracellular messenger substances cAMP / cGMP and IP3. Coupling paths can also be recorded, as a result of which the messenger concentration - for example of cAMP / cGMP - drops.
  • the availability of the receptors in a stably expressed form offers the possibility of examining defined, homogeneous receptor preparations.
  • the cells can be cultivated in small dishes, so that many substances can be tested in parallel. This option is particularly important for screening procedures in the pharmaceutical industry.
  • the examination method according to the invention offers a great time advantage.
  • the change in the intracellular Ca 2+ concentration can be registered and quantified within a few minutes.
  • the process can be largely automated and many work steps can be carried out mechanically.
  • the method of low-stringent hybridization of gene libraries was used to isolate the receptor gene.
  • a Hindlll / Nrul restriction fragment of the Drosophila dopamine Dl receptor (DmDop [-126]; Gotzes, F. and Baumann, A .: Functional properties of Drosophila dopamine Dl receptors are not altered by the size of the N-terminus ; Biochemical Biophysical Research Communications 222, 1996, 121-126).
  • the plasmid DNA was cleaved as follows:
  • the incubation was carried out at 37 ° C. for 90 min.
  • the mixture was then mixed with 6 ⁇ l stop buffer (5 ⁇ ) (100 mM EDTA, 20% (w / v) Ficoll 400, 0.01% (w / v) bromophenol blue , 0.01% (w / v) Xylencyanol) added, applied to a 0.75% agarose gel (with ethidium bromide) and separated electrophoretically.
  • the -1000 bp HindIII / Nrul fragment was cut out of the agarose gel under UV light and the DNA from the agarose piece was centrifuged (Heery, DM et al .: A simple method for subcloning DNA fragments from gel slices; Trends in Genetics 6, 1990, 173). 100 ng of the purified fragment were radioactively labeled with [32p] dCTP using the "Megaprime DNA Labeling System" from Amersham.
  • Hybridization solution was exchanged, which contained ⁇ 1 x 10 ⁇ cpm / ml of the radiolabelled sample.
  • the first hybridization was carried out under high stringency for 16 h at 65 ° C.
  • the non-specifically bound radioactivity was removed by two washing steps at 65 ° C in 1 x SET / 0.1% SDS for 30 minutes each.
  • Specifically bound radioactivity was visualized by placing an X-ray film for 4-14 h at -80 ° C. The signals found correspond to gene sequences which are identical to the DmDopl gene. 33973
  • the stringency was reduced in a second round of hybridization.
  • the hybridization temperature was reduced from 65 ° C to 52 ° C.
  • the non-specifically bound radioactivity was removed in two washing steps at 52 ° C. in 2 ⁇ SET / 0.1% SDS for 25 minutes each.
  • the specific signals were again made visible by placing an X-ray film. In comparison to the first round of hybridization, an additional signal was identified on the X-ray film.
  • the signal was assigned to a "plaque region" on the agar plate. This region was excised from the agar and eluted in phage buffer (Sambrook, J. et al., See above). After several rounds of separation, the hybridization signals could finally be assigned to individual plaque regions on the agar plate. The recombinant ⁇ phage DNA was then isolated from small lysates
  • the incubation was carried out at room temperature for 3 h.
  • the ligation mixture was transformed into transformation-competent cells of the bacterial strain XL1-Blue (Invitrogen).
  • the cells were spread on LB agar plates containing ampicillin (Sambrook, J. et al, see above) and incubated at 37 ° C. overnight.
  • Individual colonies were grown in 5 ml LB liquid cultures and the plasmid DNA was isolated by the alkaline lysis method (Sambrook, J. et al., See above). The correct incorporation of the DNA fragments was checked by restriction analyzes. Larger amounts of the plasmid subclones were purified from 50 ml liquid cultures.
  • the nucleic acid sequence of the subcloned DNA fragments was determined by the chain termination method (Sanger, F. et al: DNA sequencing with chain-terminating inhibitors; Proceedings National Academy Sciences USA 74, 1977, 5463-5467).
  • a comparison of the nucleic acid and derived amino acid sequences with those of the DmDopl receptor showed that the cloned genomic DNA contains gene sequences which code for two previously unknown members of the G protein-coupled receptor gene family.
  • the new sequence information was used to synthesize oligonucleotides that were used in polymerase chain reactions (PCR).
  • the 5 'oligonucleotide had the sequence: (# 1) 5'-GCCGTACTCGAGTTCATCAAC-3'. Two 3 'located
  • PCR approaches were carried out with the oligonucleotide pairs # 1 / # 2 and # 1 / # 3.
  • a 2nd strand cDNA preparation which had been synthesized on Drosophila head mRNA as starting material, served as the template.
  • the PCR approaches had the following composition:
  • the amplification was carried out with the following parameters:
  • the PCR fragments from the subclones were cut out with EcoRI / HindIII, separated by gel electrophoresis and isolated from the agarose (see above). The fragments were radioactively labeled (see above) and used to hybridize an adult Drosophila head cDNA library.
  • the library has already been used to isolate the DmDopl receptor gene (Gotzes, F. et al., See above).
  • the cDNA molecules were cloned into ⁇ gtll phages. The library was hybridized with high stringency (see above).
  • the DNA of the individual phages was prepared from small lysates (Santos, M.A., see above), cut with EcoRI and subcloned into a pBluescript vector which was also cut with EcoRI.
  • the nucleic acid sequences of the recombinants were determined in an overlapping double-stranded manner using suitable subclones and gene-specific oligonucleotides according to the chain termination method (Sanger, F. et al., See above).
  • the octopamine receptor genes should be expressed in a human embryonic kidney cell line (HEK 293 cells).
  • HEK 293 cells human embryonic kidney cell line
  • CCACC consensus sequence of the ribosome binding site of vertebrate gene sequences
  • PCR polymerase chain reaction
  • the mutagenesis oligonucleotide had the sequence: 5'-CTCAGGAATTCCACCATGAATGAAACAGAGTGC-3 '.
  • the counter oligonucleotide for the PCR has the sequence: 5'-CCATCCTCCGAGCTTGA-3 '. This sequence is complementary to nucleotides 1009-1025 of the open reading frame of the nucleic acid sequence of the Drosophila octopamine receptors.
  • the PCR was carried out in a 100 ⁇ l batch:
  • Receptor cDNA clone in pBluescript, 10 ng
  • mutagenesis oligonucleotide 10 ng / base x ul
  • the amplification was carried out with the following parameters: 1 cycle: 94 ° C, 2 min. 30 sec.
  • the fragment was extracted twice with phenol / chloroform, then twice with chloroform and then precipitated by adding 0.1 vol. LiCl (3 M) and 3 vol. Ethanol (absolute). After centrifugation (Sigma centrifuge, 18,000 x g, 10 min., 4 ° C.) the precipitate was dried and taken up in 10 ⁇ l TE buffer (10 mM Tris-HC1, pH 7.4; 1 mM EDTA).
  • the fragment was cut with the restriction enzymes EcoRI (5 'end) and Nael (cleaves at position 426):
  • the incubation was carried out at 37 ° C. for 90 min.
  • the mixture was then mixed with 5 ⁇ l stop buffer (5 ⁇ ) (100 mM EDTA, 20% (w / v) Ficoll 400, 0.01% (w / v) Bromophenol blue, 0.01% (w / v) xylene cyanol) added, applied to a 1.5% agarose gel (with ethidium bromide) and separated electrophoretically.
  • the -440 bp EcoRI / ael fragment was cut out of the agarose gel under UV light and the DNA from the piece of agarose was centrifuged (Heery, DM et al .: A simple method for subcloning DNA fragments from gel slices; Trends in Genetics 6, 1990, 173).
  • the complete cDNA clone in pBluescript was also restricted with EcoRI and Nael.
  • the incubation was carried out at room temperature for 3 h.
  • the ligation approach was carried out in transformation-competent cells of the bacterial strain MC1061-P3 (Invitrogen) transformed.
  • the cells were plated on ampicillin containing LB agar plates (Sambrook, J. et al: Molecular cloning: A laboratory manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and plated overnight at 37 ° C incubated. Individual colonies were grown in 5 ml LB liquid cultures and the plasmid DNA was isolated by the alkaline lysis method (Sambrook, J. et al, see above).
  • the correct incorporation of the receptor-coding cDNA was checked by a restriction analysis with the restriction enzyme BamHI. This creates two BamHI fragments of 684 bp and 1395 bp in size.
  • the nucleic acid sequence of the PCR-amplified region was additionally determined from two clones into which the cDNA was correctly incorporated. After this test, larger amounts of the pcDNAI construct (pcDmOCTIB) were purified from 250 ml liquid cultures.
  • the gene fragment was cut out of the pcDNAI construct (pcDmOCTIB) again.
  • the restriction enzymes Hindlll and Xbal were used for this. These enzymes cut only in the vector sequence (in the area of the multiple cloning site, MCS), but not within the receptor sequence.
  • the cDNA fragment was isolated as described above and then ligated into a pcDNAINeo vector (Invitrogen), which was also cut in HindIII / Xbal.
  • the ligation batch was transformed into MC1061-P3 bacterial cells and spread on LB agar plates containing ampicillin.
  • the cells were loaded with the fluorescent dye FURA-2 1 h before the experiment (Frings, S. et al: Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels; Neuron 15, 1995, 169-179).
  • the measurement was then carried out in the Ca 2+ imaging apparatus.
  • a glass plate was transferred to the measuring chamber and washed with PBS.
  • the PBS was exchanged for a PBS solution using a perfusion device defined concentration of the ligand to be tested, in the present case 1 ⁇ M octopamine.
  • the methodical approach is comparable to the transient transfection procedure.
  • the pcNeoDmOCTIB construct was used for the transfection.
  • the neomycin (neo) resistance gene is still present on this plasmid, the expression of which enables the selection of cells which have taken up the plasmid, since they are resistant to the addition of the antibiotic G418 (geneticin). Cells that have not taken up a plasmid die after a few days when G418 is added to the medium.
  • 10 ⁇ g of the pcDNAINeoDmOCTlB construct were transfected with the calcium-phosphate method (Chen, C. and Okayama, H., see above) in approx.
  • the pharmacological properties of the cloned octopamine receptors were determined on the stable cell lines. For this purpose, cells were sown on poly-L-lysine-coated flakes (see above) and transferred to the Ca2 + imaging apparatus. The cells reacted to the addition of 1 ⁇ M concentrations of the neurotransmitters tyramine and octopamine with a significant increase in the intracellular Ca2 + concentration. The Ca2 + was not released continuously but oscillatingly. No increase in the intracellular Ca2 + concentration was observed when incubated with the neurotransmitters dopamine and serotonin.
  • Octopamine causes a significant increase in the intracellular Ca2 + concentration.
  • the reaction to the incubation takes place with
  • the incubation with the antagonist can be started both before and after the addition of the octopamine. In both

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Abstract

L'invention concerne un procédé permettant de préparer des cellules produisant un récepteur d'octopamine. A cette fin, au moins un gène codant un récepteur d'octopamine est transformé en cellule hôte. Le clonage de gènes récepteurs à neuromédiateurs et leur expression stable permettent d'examiner, pour la première fois, des sous-types définis de récepteurs à neuromédiateurs. Les cellules transformées par le gène récepteur à neuromédiateurs s'utilisent dans le cadre d'un procédé simple d'examen des propriétés de récepteurs à neuromédiateurs. Selon ce procédé, on fait agir un ligand à examiner sur ces cellules et l'effet produit est déterminé par analyse des substances messagères intracellulaires.
PCT/EP1998/008411 1997-12-23 1998-12-22 Cellules clonees produisant un recepteur d'octopamine WO1999033973A2 (fr)

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DE19757695.8 1997-12-23
DE1997157695 DE19757695A1 (de) 1997-12-23 1997-12-23 Klonierte, Neurotransmitter-Rezeptor produzierende Zellen

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WO1999033973A2 true WO1999033973A2 (fr) 1999-07-08
WO1999033973A3 WO1999033973A3 (fr) 1999-09-16

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7541155B2 (en) 2003-04-24 2009-06-02 Tyratech, Inc. Methods of screening compositions for potential insect control activity
US8865230B2 (en) 2006-06-27 2014-10-21 Tyratech, Inc. Compositions and methods for treating parasitic infections

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10150971B8 (de) * 2001-10-05 2006-11-09 Technische Universität Dresden Verfahren und Vorrichtung zur Messung der Rezeptoraktivität an transfizierten Zellen
US7622269B2 (en) 2004-03-19 2009-11-24 Tyratech, Inc. Methods of screening tyramine- and octopamine-expressing cells for compounds and compositions having potential insect control activity
MX2009000548A (es) 2006-07-17 2009-05-28 Tyratech Inc Composiciones y metodos para controlar insectos.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344776A (en) * 1991-03-28 1994-09-06 The United States Of America As Represented By The Department Of Health And Human Services DNA encoding an insect octopamine receptor
WO1999021891A1 (fr) * 1997-10-27 1999-05-06 Baylor College Of Medicine Recepteur d'octopamine d'invertebres

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7541155B2 (en) 2003-04-24 2009-06-02 Tyratech, Inc. Methods of screening compositions for potential insect control activity
US8507013B2 (en) 2003-04-24 2013-08-13 Tyratech, Inc. Compositions for controlling insects
US8865230B2 (en) 2006-06-27 2014-10-21 Tyratech, Inc. Compositions and methods for treating parasitic infections

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