WO2004017728A2 - Procedes - Google Patents

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Publication number
WO2004017728A2
WO2004017728A2 PCT/GB2003/003647 GB0303647W WO2004017728A2 WO 2004017728 A2 WO2004017728 A2 WO 2004017728A2 GB 0303647 W GB0303647 W GB 0303647W WO 2004017728 A2 WO2004017728 A2 WO 2004017728A2
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Prior art keywords
insect
gene
expression
transgenic
dna
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PCT/GB2003/003647
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English (en)
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WO2004017728A3 (fr
Inventor
Sheetal Mansukhlal Shah
Robert John Lind
John David Windass
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Syngenta Limited
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Priority to AU2003264722A priority Critical patent/AU2003264722A1/en
Publication of WO2004017728A2 publication Critical patent/WO2004017728A2/fr
Publication of WO2004017728A3 publication Critical patent/WO2004017728A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
    • A01K2217/058—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to methods of screening for potential insecticidal agents using genes encoding insect P450 enzymes.
  • the invention relates to methods of using insect cyp genes and/or their promoters, as well as the products of said cyp genes, to identify and/or optimise potential insecticidal agents.
  • the methods employ .transgenic cells and or whole organisms in which one or more cyp gene is deleted or under-expressed as well as transgenic cells, cell-lines and/or organisms that over-express one or more cyp genes or express one or more cyp genes in a heterologous manner.
  • the invention further extends to the transgemc cells, cell-lines and/or organisms used in the methods of the invention as well as to DNA constructs comprising one or more cyp gene and DNA constructs comprising the promoter of a cyp gene.
  • Cytochrome P450 monoxygenases comprise a superfamily of enzymes that are found in varying numbers in almost all living organisms from bacteria to mammals. These enzymes are known to catalyse a variety of different types of chemical reaction, including for example aromatic and aliphatic oxidation, hydroxylation, dealkylation, epoxidation, oxidative deamination, desulphuration and dehalogenation reactions. In addition to monoxygenase activity, the P450s also demonstrate reductase, peroxidase and oxidase activities. It is thus unsurprising that, given their wide range of catalytic activities, these enzymes have been shown to be involved in the metabolism of a wide range of xenobiotics.
  • One conventional method of identifying novel insecticidal compounds relies on screening compounds against wild-type insects and assessing whether the compound has any deleterious effect on the insect.
  • potential insecticidal compounds are screened for activity against wild-type insects, only very few are shown to be highly insecticidally active and it is routinely only these compounds that are taken forward into further development as a potential insecticide.
  • the vast majority of compounds, which either exhibit only a low level of insecticidal activity or which have no insecticidal activity at all, are often discarded.
  • the apparent lack of insecticidal activity of a compound screened against wild- type insects may be due to the fact that the compound is truly incapable of acting as an insecticide.
  • the apparent lack of insecticidal activity may be due to the compound being metabolised by the insect. It is thought that some insect cyp genes (in particular cyp ⁇ genes of Drosophila and Musca species; Maitra et al. supra) are constirutively expressed at a low level in the wild-type insect. Thus the low level of expression of particular P450 genes and concomitant low level of residual activity of the P450 enzymes coded therefore could be contributing to the apparent lack of insecticidal activity of a compound.
  • a transgenic insect or insect cell wherein the level of expression of a P450 gene has been reduced relative to the level of expression of said P450 gene in a non-transgenic insect or insect cell.
  • transgenic insect or insect cell of the invention does not include a naturally occurring or chemically produced mutant insect or insect cell wherein the level of expression of a P450 gene has been reduced relative to the level of expression of said P450 gene in a non- mutant or wild-type insect or insect cell.
  • the P450 gene whose level of expression is reduced in a transgenic insect or insect cell of the invention is one of the 90 cyp genes found in D. melanogaster (i.e.
  • the nucleotide sequences encoding the above cyp genes may be obtained from the FlyBase database, which is a database of genetic and molecular data for Drosophila.
  • the database is maintained by a consortium of researchers funded by the National Institutes of Health, U.S.A., and the Medical Research Council, London and the database maybe accessed at: http://flybase.bio.indiana.edu/.
  • the P450 gene is selected from the group consisting of the cyp ⁇ gene family from D. melanogaster and homologues thereof.
  • the cyp ⁇ genes from D. melanogaster include: cyp ⁇ vl, cyp ⁇ tl, cyp ⁇ al ⁇ , cyp ⁇ wl, cyp ⁇ a2, cyp ⁇ ul, cyp ⁇ al4, cyp ⁇ alS, cyp ⁇ al3, cyp ⁇ gl, cyp ⁇ g2, cyp ⁇ t3, cyp ⁇ a22, cyp ⁇ al7, cyp ⁇ al9, cyp ⁇ a9, cyp ⁇ a20, cyp ⁇ a21, cyp ⁇ a8, cyp ⁇ d2, cyp ⁇ dS, cyp ⁇ d4, cyp ⁇ al8 and cyp ⁇ a23.
  • the P450 gene is selected from the group consisting of cyp ⁇ gl, cyp ⁇ a2, cyp ⁇ a8 and homologues thereof, and it is particularly preferred that the P450 gene is either cyp ⁇ gl or cyp ⁇ a2.
  • homologue refers to any gene which exhibits substantial identity to a named P450 or cyp gene.
  • a homologue exhibits at least 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% identity with a named P450 or cyp gene.
  • Most preferably a homologue exhibits at least 96%, 97%, 98% or 99% identity with a named P450 or cyp gene.
  • Homologues include P450 or cyp genes found in the same genus and species as the named P450 or cyp gene, as well as P450 or cyp genes from insects of other genera and/or species.
  • the homologue may come from the group of insects consisting of: Drosophila sp.
  • the homologue will come from a known insect pest species, including in particular (but not limited to) Heliothis virescens and Manduca sexta.
  • the transgenic insect or transgenic insect cell may be any insect or insect cell which is capable of being transformed.
  • the transgenic insect may be, or the transgenic insect cell may come from, any one of the following insects: Drosophila sp. Bombyx mori, Tribolium castaneum, Aedes aegyptii, Anopheles gambiae, Anopheles albimanus, Anopheles stephensi, Ceratitis capitata, Pectinophora gossypiella,, Helicoverpa zea, Bactrocera dorsalis, Anastrepha suspense, Musca domestica, Stomoxys calcitrans, and Lucilia cuprina.
  • a transgenic insect cell of the invention may also be derived from Spodoptera frugiperda or Trichoplusia ni. In one embodiment it is particularly preferred that the transgenic insect cell is derived from a D. melanogaster S2 cell.
  • transgenic insects or insect cells of the invention are derived from D. melanogaster.
  • Transgenic insects and insect cells according to this aspect of the invention exhibit a reduced level of expression of a P450 gene in comparison to the level of expression of said P450 gene in a wild-type non-transgenic insect or insect cell.
  • the transgenic insect is D. melanogaster
  • D. melanogaster Canton S is particularly suitable as a wild- type non-transgenic insect and where the transgemc insect cell is derived from a
  • Drosophila cell line such as the Drosophila S2 cell line
  • the untransformed cell line may act as a suitable non-transgenic insect cell for the purposes of comparison.
  • a reduced level of expression of a P450 gene includes any level of expression that is less than the level of expression of the P450 gene in the non-transgenic insect or insect cell and includes no detectable expression of said P450 gene in the transgenic insect or insect cell.
  • the reduced level of expression of the P450 gene in the transgenic insect or insect cell may be effected through gene disruption at the locus for the P450 gene in the insect concerned. This may achieved through homologous recombination (see Rong & Golic 2000 Science 288(5473):2013-2018 for methodology relating to homologous recombination in Drosophila) or through insertional mutagenesis e.g. transposon insertion.
  • the reduced level of P450 expression may be effected using an RNA- interference (RNAi) mediated knockdown or knockout of P450 or cyp gene function, via a either a transient RNAi approach, or via the generation of a stable transgenic insect line (Kennerdell& Carthew 2000, Nature Biotech. 17:896-898. Where a stable Drosophila line is produced, it is preferred that the reduction in P450 gene expression level is inducible. This may be achieved by employing the GAL4-UAS system (Brand & Perrimon, 1993, Development 118:401-415) as described herein in the Examples.
  • GAL4-UAS system Brand & Perrimon, 1993, Development 118:401-415
  • an insect or insect cell is transformed with a DNA construct comprising a first DNA sequence encoding a fragment of an insect P450 gene and a second DNA sequence which is the reverse complement of the first DNA sequence, wherein the first and second DNA sequences are present on the same strand of DNA.
  • a mRNA molecule will be produced that is capable of forming a hairpin or stem-loop structure by virtue of the complementarity of the regions in the mRNA which correspond to the first and second DNA sequences.
  • This double stranded mRNA region acts as a source of short interfering RNA molecules and thus mediates the knockdown or knockout of P450 gene function, which is manifested in transgenic insects or insect cells as a reduced level of expression of the P450 or cyp gene.
  • the invention provides a DNA construct comprising a first DNA sequence encoding a fragment of an insect P450 gene and a second DNA sequence which is the reverse complement of the first DNA sequence, wherein the first and second DNA sequences are present on the same strand of DNA and are operably linked to a promoter region and optionally a terminator region.
  • the minimum size of a short interfering RNA is between 21 and 23 nucleotides (Zamore et al. 2000 Cell 101:25-33). Accordingly for just a single short interfering RNA to be produced from the transcript of a DNA construct of this aspect of the invention, the minimum length of the first and second DNA sequences in said DNA construct is 23 nucleotides. However, it is preferred that the length of the first and second DNA sequences is considerably longer than 23 nucleotides. Preferably the first and second DNA sequences will be greater than or equal to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1500 nucleotides in length.
  • a DNA construct of the invention will additionally comprise a region of DNA which acts as a spacer region between the first and second DNA sequences.
  • a DNA sequence may be incorporated to facilitate cloning or to facilitate the formation of a double-stranded mRNA structure, such as a hairpin or stem-loop structure, when the DNA sequence of the DNA construct is transcribed.
  • Sequences that are known to be introns in a gene of the insect that is to be transformed with a DNA construct of the invention are particularly suitable for this purpose.
  • the first and second DNA sequences that are present in a DNA construct of the invention are operably linked to a promoter region.
  • the promoter region will be any promoter region capable of driving expression of the first and second DNA sequences in the insect or insect cell into which a DNA construct of the invention is to be introduced.
  • the promoter region may comprise a constitutive promoter, a tissue- or developmentally-specific promoter, or an inducible promoter.
  • the promoter region comprises an inducible promoter so that the reduction in the level of expression of the P450 gene is controllable.
  • the inducible promoter comprises UAS hsp70 TATA promoter sequences responsive to the GAL4 protein.
  • DNA constructs of the invention optionally comprise a transcriptional terminator region.
  • such a transcriptional terminator region will be one that is suitable for the termination transcription in the insect/insect cell into which the DNA construct is to be introduced.
  • Transcriptional terminator regions suitable for this purpose are described in the art.
  • DNA constructs of the invention are introduced into insect cells to form transgenic insects and insect cells using any suitable method available in the art.
  • D. melanogaster embryos may be transformed using the methodology described by Spradling & Rubin 1982 (Science 218:341-347), for example, as described herein in the Examples.
  • the promoter region of a DNA construct of the invention comprises a constitutive promoter region, transformation of the DNA construct into an insect or insect cell will yield a transgenic insect or insect cell of the invention.
  • a transgenic insect or insect cell of the invention may be produced merely by fransforrning an appropriate insect of insect cell with a DNA construct as described hereinbefore.
  • the promoter region comprises an inducible promoter an inducing agent must be applied to (or be present within) the transformed insect or insect cell.
  • a DNA construct of the invention is introduced into a first insect and a second DNA construct, which encodes a protein capable of regulating expression from the inducible promoter in the DNA construct of the invention, is introduced into a second insect.
  • the first and second insects are then genetically crossed to yield progeny, a proportion of which will comprise both the first and second DNA constructs.
  • the population of progeny comprising both the first and second DNA constructs will comprise not only a DNA construct of the invention but also the inducing agent necessary to effect induction from the promoter region in the first DNA construct. Consequently the population of progeny, which comprises both the first and second DNA constructs, consists of transgenic insects of the invention.
  • a method of producing a transgemc insect of the invention which comprises: (i) fransforrning a first insect with a first DNA construct as described hereinbefore wherein the promoter region comprises an inducible promoter; (ii) transforming a second insect with a second DNA construct encoding a protein that is capable of inducing expression from the inducible promoter in the first DNA construct; (iii) crossing the transformed insects resulting from steps (i) and (ii) to obtain progeny; wherein a population of the progeny resulting from step (iii) comprises both the first and the second DNA constructs and when the protein encoded in the second DNA construct is expressed, the level of expression of the P450 gene is reduced in said population of the progeny relative to the level of expression of the P450 gene in a non-transgenic insect.
  • Insects containing the second DNA construct e.g. insects already transformed with a DNA construct encoding a protein that is capable of inducing expression from the inducible promoter in the first DNA construct are also readily available in the art (see Example 3 hereinafter).
  • the invention also provides a method of producing a transgenic insect of the invention, which comprises: (i) transforming a first insect with a first DNA construct as described hereinbefore wherein the promoter region comprises an inducible promoter; (ii) crossing the transformed insect resulting from step (i) with an insect that expresses an inducing protein that is capable of inducing expression from the inducible promoter in the first DNA construct, to obtain progeny; wherein a population of the progeny resulting from step (ii) comprise the first DNA construct and express the inducing protein so that the level of expression of the P450 gene is reduced in said population of the progeny relative to the level of expression of the P450 gene in a non- transgenic insect.
  • a transgenic insect cell may be made by co-transforming a suitable insect cell with a DNA construct of the invention and a second DNA construct that encodes a protein capable of regulating expression from the inducible promoter in the DNA construct of the invention.
  • transgenic insects and transgenic insect cells of the invention are more susceptible to compounds that are subject to metabolism by the product of the P450 gene whose expression level has been reduced therein.
  • the transgenic insects and insect cells of the invention are useful in identifying compounds which are potential insecticides and which would not have been identified, or would have only been identified as being weakly active, in a conventional screen using a wild-type and/or non-transgenic insect or insect cell.
  • a method of identifying or verifying the ability of a compound to act as an insecticide comprises: (i) placing a transgenic insect or insect cell of the invention in contact with the compound; (ii) assessing the transgenic insect or insect cell that has been placed in contact with the compound in step (i) for any deleterious effect on the insect or insect cell; wherein the presence of a deleterious effect is indicative that said compound is capable of acting as an insecticide.
  • a deleterious effect may be manifested as the death of the insect or insect cells, or by slow growth of the insect or insect cell in comparison to insect or insect cells that have not been treated with the compound, or by some other clear phenotypic change in treated insects or insect cells in comparison to untreated insects or insect cells.
  • the present invention also provides assays for identifying insecticidal compounds that are resistant to degradation by one or more insect P450 gene product, in particular compounds that are resistant to degradation by the products of insect cyp genes.
  • Particular cyp genes of interest are the cyp ⁇ genes as described above.
  • the cyp ⁇ genes include the cyp ⁇ gl, cyp ⁇ a2 and cyp6A8 genes, and the use of these genes is considered as particularly desirable.
  • the invention provides a method of reducing the susceptibility of a compound to degradation by one or more cyp gene products.
  • assays utilise transgenic insect and insect cells as described hereinbefore as well as cells and/or whole organisms that over-express an insect P450 or cyp gene, or which express an insect P450 or cyp genes in a heterologous manner.
  • cells and/or whole organisms that over- express an insect P450 or cyp gene, or which express an insect P450 or cyp gene in a heterologous manner
  • cells and/or whole organisms may comprise a DNA construct comprising a cyp gene of Drosophila melanogaster, in particular one of the 90 known cyp genes listed hereinbefore, operably linked to a promoter region and optionally a transcriptional terminator.
  • expression constructs are referred to herein as "expression constructs”. It is preferred that the cyp gene will be selected from the group consisting of cyp ⁇ gl, cyp ⁇ a2, cyp ⁇ a8 and homologues thereof.
  • the cyp gene is cyp ⁇ gl or cyp ⁇ a2.
  • the promoter region to which the cyp gene is operably linked will be any promoter region capable of driving expression of the cyp gene in the host cell into which an expression construct comprising the cyp gene is to be introduced.
  • the promoter will be operable in that bacterial cell.
  • the promoter will be operable in that yeast cell and the same logic prevails if the construct is to be introduced into an insect cell or a mammalian cell.
  • the promoter region may thus be the native promoter that is associated with the individual cyp gene in nature, provided that the promoter is capable of driving expression in the host cell into which the construct is to be introduced.
  • the promoter region may be any one of those with which the skilled man is familiar with for mediating expression in the host of choice.
  • the promoter region may comprise a constitutive promoter, a tissue- or developmentally-specific promoter, or an inducible promoter.
  • the promoter region will comprise the polyhedrin promoter from the Autographa calif ornica nuclear polyhedrosis virus.
  • Expression constructs according to this aspect of the invention optionally comprise a transcriptional terminator region.
  • a transcriptional terminator region will be one that is suitable for termination transcription in the host cell into which the expression construct is to be introduced.
  • Transcriptional terminator regions suitable for this purpose are described in the art.
  • the present invention also provides a cell transformed with an expression construct as described hereinbefore.
  • Suitable host cells for transformation with a DNA construct of the invention include bacterial cells (such as Esche ⁇ chia coli), yeast cells (for example, Saccharomyces cerevisiae, Pichia species, Schizosaccharomyces pombe), insect cells (for example, Drosophila S2 cells, Spodopterafrugiperda Sf9 or Sf21 cells, Trichoplusia ni High FiveTM cells), and mammalian cells.
  • an expression construct of the invention is transformed into insect cells, in particular Sf9 cells.
  • Introduction of the expression construct into a host cell may employ any suitable technique.
  • eukaryotic cells such techniques include calcium phosphate transfection, DEAE-Dextran, electroporation, particle bombardment, liposome-mediated transfection or transduction using retrovirus, adenovirus or other viruses, such as vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation or transfection using bacteriophage.
  • Sf9 cells are transformed using baculovirus, using for example, the protocols supplied with the MaxBac®2.0 -Complete Baculovirus Expression System (Invitrogen).
  • the expression construct may also be introduced into a whole organism i.e.
  • transgenic Drosophila insects may be produced wherein the level of expression of the P450 gene present in the expression construct is amplified over the level of expression observed in wild-type non-transformed Drosophila.
  • Cells or insects transformed with an expression construct may be employed in a method of assaying the susceptibility of a compound to degradation specifically by the product of the cyp gene present in the expression construct.
  • the invention also provides a method of assaying the susceptibility of a compound to degradation specifically by the P450 or cyp gene encoded on the expression construct which comprises: a) culturing in a suitable growth medium a population of cells or an insect transformed with a DNA construct of the invention b) incubating said compound with said population of cells or an insect; and c) determining after said incubation the level of one or more degradation products of said compound in said population of cells or said insect; wherein the presence of a measurable level of one or more degradation products is indicative that said compound is susceptible to degradation by the product of the cyp gene present in the expression construct.
  • the suitable growth medium will be dependent upon the type of transformed cells of the invention.
  • the transformed cells of the invention are Saccharomyces cerevisiae yeast cells, YPD is one such suitable medium.
  • the transformed cells are Sf9 or Sf21 cells,
  • Grace's Insect Cell Culture Medium is one such medium.
  • suitable culture medium is Schneider's insect medium, optionally supplemented with foetal calf serum. The skilled man will be familiar with alternative media that are suitable for growth of the particular host cells chosen.
  • Degradation products of the compound being assayed may be measured using any suitable analytical technique.
  • LC-MS is the technique employed to detect and measure metabolites.
  • the cells or insects treated with a compound may be lysed and the cell contents released into a suitable buffer.
  • suitable methods for cell lysis which are well-documented in the art.
  • the susceptibility of that compound to degradation by that cyp gene may be assessed.
  • Such a test allows better detection of weak insecticidally active compounds that may otherwise not have been identified, since they are susceptible to degradation by P450s and also provides confirmation that the degradation to which they are susceptible is mediated by the gene product of the under- or over-expressed cyp gene.
  • the in vivo metabolism of the compound may then be examined using any suitable analytical technique (for example, an LC-MS technique) and solutions proposed for the enhancement of stability of the compound by altering the chemistry of those parts of the molecule that are subject to degradation.
  • any suitable analytical technique for example, an LC-MS technique
  • trasnsgenic insects, insect and other cells in this way forms yet another aspect of the invention.
  • a compound that has been identified or verified as being capable of acting as an insecticide using a transgenic insect or insect cell as described in the first aspect of the invention may also be tested as described above to assess whether it is susceptible to degradation by the product of the cyp gene whose expression level was reduced in the transgenic insect or insect cell against which it has been tested. It will be appreciated by the skilled man that where a compound is tested in this manner, the same cyp gene will be used in each assay. It will also be appreciated that the assay for the susceptibility to degradation may be performed initially, and the assay using a transgenic insect or insect cell as described in the first aspect of the invention may be carried out subsequently.
  • the presence of degradation products in the first assay should correlate with the observation of insecticidal activity in the second assay.
  • the absence of degradation products in the first assay need not necessarily correlate with an absence of insecticidal activity in the second assay. In such a situation a likely explanation is provided by the cyp gene or gene product being involved in the indirect mediation of metabolism.
  • the assay may be used in an iterative manner to aid the design of compounds that are less susceptible to degradation. For example, if a potential insecticide comprises a backbone chemical structure to which one or more sub-groups are attached, it may be found that by substituting one or more of such sub groups for a different sub-group and re-testing the modified compound, the modified compound may be found to have an improved resistance to degradation. If this is the case, the process may be repeated either altering the same, or a different, sub-group and then re-testing the compound in the assay(s) of the invention.
  • a method of identifying or verifying the ability of a compound to act as an insecticide which comprises: a) culturing in a suitable growth medium a population of insect cells transformed with an expression construct of the invention; b) incubating said compound with said population of cells; and c) determining after said incubation the viability of said cells; wherein a lack of viability of said cells is indicative that said compound is capable of acing as an insecticide.
  • a lack of viability of cells is preferably indicated by cell death.
  • the slow growth of cells treated with the compound in comparison to cells that have not been treated with the compound, or some other clear phenotypic change in treated cells in comparison to untreated cells may also be used as a measure of cell viability.
  • This aspect of the invention may be modified to identify or verify the ability of a compound to act as a pro-insecticide. Where this is the desired intention, cells transformed with an expression construct will be treated with a test compound, cell viability will be monitored post-treatment and after a period of incubation with the test compound, a sample of cells will be assayed to determine the presence or absence of metabolites of the test compound.
  • control cells non- transformed cells or cells transformed with a control DNA construct lacking the cyp gene
  • test compound and cell viability determined post treatment.
  • Compounds that have no effect on the viability of the control cells, but cause a loss in viability of cells of the invention and which are shown to be degraded by the product of the cyp gene expressed in the cells are thus identified or verified as pro-insecticidal compounds.
  • a still further aspect of the invention a method of identifying a compound that regulates expression of an insect P450 gene, which comprises: (i) culturing in a suitable growth medium a population of cells transformed with a DNA construct comprising a reporter gene operably linked to the promoter region of the P450 gene; (ii) incubating said compound with a population of cells according to step (i);
  • step (iii) determining the level of expression of said reporter gene in cells that have been incubated with said compound; and (iv) determining the level of expression of said reporter gene in a population of cells according to step (i); wherein, a difference in the levels of reporter gene expression determined at (iii) and (iv) is indicative that said compound is capable of acting as a regulator of expression of said P450 gene.
  • reporter gene constructs DNA constructs for use in this aspect of the invention are referred to herein as "reporter gene constructs" and will comprise any suitable reporter gene available in the art, for example the reporter gene may be a gene encoding : ⁇ -galactosidase, luciferase, or a fluorescent protein, such as for example green fluorescent protein.operably.
  • the reporter gene will be operably linked to the promoter region of an insect P450 gene.
  • the promoter region will comprise the promoter of one of the 90 known cyp genes from D. melanogaster as listed above. Even more preferably the promoter region will comprise the promoter of the cyp ⁇ gl gene or the cyp ⁇ a2 gene. In a preferred embodiment, the promoter region will lack the consensus sequences for phenobarbitol induction (i.e. the promoter will lack so called "barbie-box" regions as defined by Shaw and Fulco 1993, J. Biol. Chem. 268:2997-3004).
  • the promoter regions of insect P450 genes for use in this aspect of the invention may be isolated and cloned using standard molecular biological techniques.
  • a reporter gene construct for use in this aspect of the invention may be transformed into Drosophila S2 cells using any suitable method available in the art. Where the reporter gene is luciferase the level of reporter gene expression may be assayed using the Luciferase Assay System from Promega.
  • Compounds that are identified according to this aspect of the invention in particular those that down-regulate expression of cyp ⁇ gl or cyp ⁇ a2 may be useful as additives to existing insecticides that are subject to degradation by the products of these two genes, or they may find utility as insecticides in their own right.
  • a compound is to be "identified” as having the ability to act as an insecticide or to act as a regulator of the expression of an insect P450 gene
  • that compound has not previously been shown to act as an insecticide or to act as a regulator of expression of an insect P450 gene.
  • any compound that has previously been shown to act in such a way is excluded from the scope of any method of "identifying" a compound, e.g. phenobarbitol which has previously been shown to regulate the expression of some cyp genes, is not considered as a compound that is capable of being “identified” as a regulator of expression of a P450 gene.
  • Figure 1 Shows a schematic representation of DNA construct
  • FIG. 3 Graphical representation of the bioassay data from the treatment of transgenic Drosophila that under express cyp ⁇ gl (lines act-gal4/hp_l and act-gal4/+; hp_2/+), wild-type Drosophila (Canton-S - control) and mutant Drosophila that over express cyp ⁇ gl (Hikone-R).
  • Example 1 Construction of vectors for use in the production of insects wherein the level of expression of a P450 gene is reduced relative to wild type insects
  • Constructs were generated that encode inverted repeats of cyp gene fragments, which on expression fold to form an RNA duplex or as referred to herein a hairpin (hp) RNA molecule, which is capable of mediating a RNA interference (RNAi) effect.
  • the constructs generated also include a spacer sequence, which serves to facilitate cloning, between the inverted repeats.
  • the expression of the inverted repeats is under control of an inducible promoter system.
  • constructs were generated using restriction-enzyme mediated cloning (1.1) and also site-specific recombinase mediated cloning (1.2).
  • genomic DNA from a wild- type lab strain Canton-S (Bloomington stock centre, stock number 1; http:/flystocls.bio.Indiana.edu, Drosophila Stock Center, Department of Biology, Indiana University, 1001 E. 3 rd St., Bllomington, IN 47405-3700, USA) was isolated using standard molecular biology techniques (Sambrook, et al. 1989, Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory, New York; Bender et al. 1983 J. Mol. Biol. 168: 17-33). This was then used as the DNA template for PCR amplification in various steps as described below.
  • the spacer sequence was cloned into the parent vector (pB-UAS w+, described in 1.1.1 below). Then the two inverted repeats of cyp gene fragments were inserted as described in 1.1.2. Two different hairpin constructs were made, these differed from each other in the size of the inverted repeat; one comprised a long gene fragment ( ⁇ 1700 bp) (1.1.2a), and the other comprised from a shorter gene fragment (-600 bp) (1.1.2b).
  • intron sequence from the cyp4e2 gene was first cloned into the vector pB-UAS w+ (Genbank AY196823) to provide the spacer sequence to facilitate cloning of inverted repeats.
  • This intron was PCR-amplified from the Canton-S genomic DNA preparation using primers cyp4e2_intron_F and cyp4e2_intron_R which contain introduced Xbal and BamHI sites, respectively, into their 5' termini.
  • the sequences of these primers are: cyp4e2_intron_F 5' GCTCTAGAGCAGATCTGTCAGGTGGGTTTTTCATAG 3' (SEQ ID NO 1).
  • cyp4e2_intron_R 5' CGGGATCCCGCATATGTATTTCTATATAGACGTGAG 3'.(SEQ ID NO 2).
  • the Xbal-BamHl fragment from this PCR product was then cloned into the corresponding Xbal-BamHl sites of the vector pB-UAS w+, using standard molecular biology techniques.
  • cyp ⁇ gl For cloning of the inverted repeat cyp ⁇ gl sequences, two constructs were generated: one that encodes a long inverted repeat (the 'cyp6gl_kmg' construct), and one that encodes a shorter inverted repeat ('cyp6gl_short' construct).
  • the cyp ⁇ gl gene sequence from EMBL:AE003823 is shown below.
  • the cyp ⁇ gl gene fragments chosen for cloning and consequent expression as hairpin RNA molecules were selected to maximise the probability that only cyp ⁇ gl mRNA was degraded via an RNA interference mechanism.
  • Suitable gene fragments may be used, and the skilled man would recognise that these can be selected by comparing the sequence of the gene of interest (in this case cyp ⁇ gl) with the sequences of other cyp genes and choosing fragments of the gene of interest that are ill-conserved amongst the series of cyp genes compared. Preferably the fragments chosen will be unique to the gene of interest.
  • the cyp ⁇ gl genomic DNA sequence may be found in the EMBL database under the reference: AE003823.
  • the region encoding cyp ⁇ gl is shown below (in the 5' to 3' orientation), with the positions of introns underlined and in lower case and the position of the start codon and termination codon of the cyp ⁇ gl coding sequence are shown in bold and underlined.
  • the cyp6gl_long construct contains inverted repeats of bases 743-2501 of the cyp ⁇ gl gene sequence from EMBL:AE003823. This sequence from bases 743-2501 is shown below (again, intron sequences are shown in lowercase and underlined).
  • cyp ⁇ gl Jong sequence SEQ ID NO 5:
  • the Cyp ⁇ gl FWD primer (cont-rining a BamHl and a Xbal restriction site introduced at the 5' terminus of the primer: 5' GCGGATCCATTCTAGAAAGTGG CGATACCCAT TACATCG 3'-SEQ ID NO 6) and the Cyp ⁇ gl _REV primer (containing a Notl and an EcoRl restriction sites introduced at the 5' terminus: 5' TAGAATTCGCGGCCGCGATC ACACAGTTCATGCGATTC 3' -SEQ ID NO 7) were used to PCR-amplify these cyp ⁇ gl sequences from the same Canton-S genomic DNA template described earlier.
  • the cyp6gl_short construct contains inverted repeats of two cyp ⁇ gl sequences that are fused together.
  • Bases 743-1162 are fused to bases 2028-2192 of the cyp ⁇ gl gene sequence from EMBL:AE003823.
  • the fusion sequence was generated by a 2-step PCR method. First the two sequences were separately PCR-amplified from the Canton-S template DNA.
  • the primer pairs used for this reaction are Cyp ⁇ gl _ FWD and Bridge_REV (5' CGTAGAACGGCTTCATGGGCATCCCTGACGAAGAACA 3 ' - SEQ ID NO 9), which PCR-amplified bases 743-1162 of the cyp ⁇ gl gene sequence from EMBL: AE003823.
  • the primer pairs used for the other reaction are Cyp ⁇ gl REV and Bridge JFWD (5' CGTCAGGGATGCCCATGAAGCCGTTCTACGACTACAC 3' - SEQ ID NO 10), which PCR-amplified bases 2028-2192 of the cyp ⁇ gl gene sequence from EMBL:AE003823.
  • equimolar amounts of the two PCR products from these reactions were mixed together and used as the template DNA for a further PCR amplification reaction.
  • PCR amplification from this mixed template using primers Cyp ⁇ gl FWD and Cyp ⁇ gl REV, generated a fusion DNA sequence: bases 743-1162 were fused to bases 2028-2192 of the cyp ⁇ gl gene. This occured because the primers Bridge FWD and Bridge REV contain complementary sequences at their 5' ends, thus permitting annealing of the two original PCR products.
  • the Xbal-Notl fragment was isolated and cloned into the corresponding Xbal-Notl sites in the plasmid generated in 1.1.1 above, using standard restriction enzyme mediated cloning methods'
  • the BamHl-EcoRl fragment from this PCR product was then cloned into the BamHl-EcoRl sites of the resulting plasmid.
  • genomic cyp ⁇ a2 gene sequence shown below was generated from sequences in the EMBL database: sequence from EMBL Accession No:AE003790 and cDNA sequence from EMBL Accession No. DMU78088. Intron sequences are shown in lower case and are underlined and the start codon and termination codon of the cyp ⁇ a2 coding sequence are shown in bold and underlined. SEQ ID NO 11:
  • primers were designed as for cyp ⁇ gl long cloning, with the corresponding restriction sites added to the 5' termini of the primers to facilitate cloning. Constructs were then generated for cyp ⁇ a2 in the same way as described above for cyp ⁇ gl.
  • the cyp6a2_long sequence cloned from bases 1306 - 2952 of the cyp ⁇ a2 gene sequence given above is: 5' CGAAAAGGGAGCAGCTACGCAAAATGTTTGTTCTAATATACCTGTTGA
  • cvp6a2_short hairpin construct For generation of the cyp6a2_short construct, primers were designed as for cyp6gl_short cloning, with the corresponding restriction sites added to the 5' termini of the FWD and REV primers to facilitate cloning. Internal 'BRIDGE' primers were also used to facilitate fusion of the two primary PCR templates, thus creating the chimeric template for the second PCR. Constructs were then generated as described above with respect to the cyp6gl_short construct.
  • the cyp6a2_short sequence that was cloned comprises bases 1306 - 1718 fused to bases 2780 — 2952 of the cyp ⁇ a2 gene sequence given above.
  • the cyp6a2_short sequence is: 5 ' CGAAAAGGGAGCAGCTACGCAAAATGTTTGTTCTAATATACCTGTTG ATCGCGATCTCCTCGCTTTTGGCCTACTTGTACC ACCGC AACTTCAACTACTGG AATCGCCGCGGCGTGCCACACGATGCTCCTCACCCACTGTATGGCAACATGGT CGGGTTCCGGAAGAACCGGGTGATGCACGACTTCTTCTACGACTACTACAAC AAGTACCGGAAGAGCGGCTTTCCCTTCGTGGGCTTTTACTTTCTGCACAAGCC GGCCGCCTTCATCGTGGACACCCAGCTGGCCAAGAACATCCTGATCAAGGAT TTCTCGAACTTTGCCGATCGTGGCCAGTTTCACAACGGGCGCGACGACCCGCT CACGCAGC
  • the insert sequences for the selected cyp genes were PCR amplified as described above, using primers containing the appropriate recombinase target site sequences to facilitate recombinase-mediated cloning into ENTRY vectors (Invitrogen). Then, in a single step, these ENTRY-derived plasmids were mixed with the pB-UGIR w+ vector, and the desired constructs were generated via recombinase-mediated cloning following the manufacturer's protocols.
  • Figure 2 shows how the cyp sequence inserts are cloned in the tail-to-tail orientation using the pB-UGIR w+ vector (the example shown is for the cyp ⁇ gl construct).
  • Embryos were injected prior to pole cell formation; thus 0-30 min old w lls embryos were collected, dechorionated and dessicated, and then lined up on glass slides covered with double-sided sticky tape to hold the embryos in place. The embryos were covered with a minimal amount of halocarbon oil, and microinjected posteriorly with the transformation construct and helper plasmid mix. The injected embryos were then incubated at 25°C, and any hatching larvae were recovered and transferred to standard diet. Adults emerging from these larvae were then crossed to w lls adults, and stable transformants were selected from their progeny (expressing the mini-w+ marker for transformation). Several independent transgenic flies per injected construct were isolated, and stocks of these transgenic flies were established. Transgenics were also confirmed by PCR amplification of sequences specific to the transformation plasmids.
  • line hp_l which is a transgenic line derived from the 'cyp6gl_short' construct
  • line hp_2 which is a transgenic line deriving from the 'cyp6gl_long' construct.
  • the transgenic flies generated in Example 2 comprise the cyp ⁇ gl- or cyp ⁇ a2- specific hairpin RNA molecules, and the expression of these hairpin molecules is under the control of an inducible promoter. Induction of hairpin expression mediates the degradation of the corresponding endogenous mRNAs through an RNA interference (RNAi) mechanism. As expression of the hairpin molecules is under control of an inducible promoter, an inducing agent is provided to effect the RNAi in these flies. This inducing agent is provided from another transgenic line by means of a genetic cross that brings together the inducing agent and the inducible fransgene (i.e. in this case the hairpin molecule) into the same individual flies.
  • RNAi RNA interference
  • the yeast GAL4 protein acts as a transcriptional regulator and thus as an inducing agent; it binds to UAS promoter sequences and activates the expression of any sequences encoded downstream of this promoter, thus rendering the expression of such sequences inducible by the GAL4 protein
  • This binary system is applied by a genetic cross, between (a) a transgenic fly line that encodes for the transcription factor (GAL4), and (b) the transgenic fly line that contains the UAS promoter encoded upstream of the cloned cyp inverted repeat sequences (e.g. a hp_l or hp_2 transgenic fly).
  • GAL4 transgenic fly line that encodes for the transcription factor
  • the transgenic fly line that contains the UAS promoter encoded upstream of the cloned cyp inverted repeat sequences e.g. a hp_l or hp_2 transgenic fly.
  • a proportion of the progeny from such a cross will express both the GAL4 protein as well as the hairpin molecule (via GAL4 regulation), and express double-stranded (ds) RNA corresponding to partial cyp sequences encoded by the hairpin (e.g partial cyp ⁇ gl or cyp ⁇ a2 sequences).
  • dsRNA molecules then mediate degradation of the endogenous mRNA transcripts for cyp ⁇ gl and cyp ⁇ a2, via an RNAi mechanism.
  • the result is that the amount of endogenous mRNA transcripts for cyp ⁇ gl and cyp ⁇ a2 are depleted. This effects a reduced level of expression of the specific cyp gene (e.g. cyp ⁇ gl or cyp ⁇ al) and the product thereof, and a knockdown or knockout of cyp gene function is thus observed.
  • the GAL4-expressing transgenic fly line used herein was obtained from the Bloomington stock centre (supra) and is described by the genotype: yflj w[*J * ;
  • transgenic fly lines comprising cyp inverted repeat regions (line hp l, which is a transgenic line derived from the e cyp6gl_short' construct and line hp_2, which is a transgenic line deriving from the 'cyp6gl_long' construct) were crossed with the GAL-4- expressing transgenic fly line.
  • the resulting FI progeny were selected for those that carried both the GAL4 and the cyp6gl_long or cyp6gl_short transgenes.
  • the FI progeny from the cross between the GAL4 and hp_l transgenic lines are referred to herein as act-gal4/hp_l flies, and similarly the FI progeny from the cross between the GAL4 and hp_2 transgenic lines are referred to herein as act-gal4/+; hp_2/+ flies.
  • act-gal4/hp_l and act-gal4/+; hp_2/+ flies had no obvious morphological phenotype and were viable.
  • the cyp ⁇ gl or cyp ⁇ a2 mRNA depletion in these FI flies was measured at the molecular level, using a Quantitative reverse transcription-polymerase chain reaction (Q- RT-PCR) to measure the amount of cyp ⁇ gl and cyp6a2 mRNA present in these flies compared to wild-type or other suitable control flies.
  • Q- RT-PCR Quantitative reverse transcription-polymerase chain reaction
  • the primers and Taqman probes used for this mRNA detection assay were:
  • the assays showed that overall cyp ⁇ gl expression was reduced (in comparison to the Canton-S wild-type flies), ranging from approximately 5-fold in the act-gal4;hp_l flies, to approximately 100-fold in the act-gal4/+; hp_2/+ flies.
  • cyp ⁇ gl expression in the population of Hikone-R was found by this method to be approximately 16-fold higher than in the Canton-S control flies.
  • the susceptibility of the transgenic D. melanogaster to known insecticides was assessed by bioassay as follows. A 5% sucrose: 1% agar mix was prepared and 0.75 mis poured into each well of a 24-well plate, and allowed to set. The test chemical (imidacloprid or fipronil) was dissolved in a suitable ethanol-acetone based solvent system to the dilutions required.
  • the dilutions used ranged from 0 to 20,000 ppm. 20 ⁇ l of a single dilution was spread uniformly on the surface of the sucrose-agar base in a well. By applying the dilution series to consecutive wells in a plate, a dose range of the chemical was created. At least 3 replicate wells of each dose were made. The control wells contained the formulation solution only, applied similarly to the surface of the sucrose-agar base.
  • the effects on the flies were monitored for up to 6 days by scoring for mortality of the flies in the wells. Any other phenotypic effects were also assessed. Comparisons between different genotypes were carried out on data from assessments that were performed contemporaneously and by the same assessor.
  • Bioassays were carried out as described above, using Hikone-R flies as the cyp ⁇ gl overexpressing strain, Canton-S as the wild-type flies expressing normal levels of cyp ⁇ gl, and act-gal4/hp_l and act-gal4/+; hp_2/+ as the cyp ⁇ gl underexpressing strains.

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Abstract

L'invention concerne un procédé de criblage d'agents insecticides potentiels au moyen de gènes codant pour des enzymes P450 d'insectes. L'invention porte notamment sur des procédés d'utilisation de gènes cyp d'insectes et/ou de leurs promoteurs pour l'identification et/ou l'optimisation d'agents insecticides potentiels. Lesdits procédés utilisent des cellules transgéniques et/ou des organismes entiers dans lesquels au moins un gène cyp est délété ou sous-exprimé.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008083598A1 (fr) * 2006-12-30 2008-07-17 Zhicheng Shen Culture transgénique sélectivement éliminée, préparation et utilisation correspondantes
CN104059931A (zh) * 2014-07-03 2014-09-24 西南大学柑桔研究所 桔小实蝇细胞色素p450基因的rna干扰载体及其构建方法和应用
CN109112117A (zh) * 2017-09-04 2019-01-01 华中农业大学 一种分离的二化螟cyp15c1基因及其编码蛋白

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040470A2 (fr) * 1997-03-07 1998-09-17 Novartis Ag Cytochrome p450 monooxygenases
WO2001092561A2 (fr) * 2000-06-02 2001-12-06 Purdue Research Foundation Procede de criblage de resistance croisee negative
WO2003025223A2 (fr) * 2001-09-20 2003-03-27 The University Of Bath Ameliorations apportees au criblage d'insecticides ou s'y rapportant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040470A2 (fr) * 1997-03-07 1998-09-17 Novartis Ag Cytochrome p450 monooxygenases
WO2001092561A2 (fr) * 2000-06-02 2001-12-06 Purdue Research Foundation Procede de criblage de resistance croisee negative
WO2003025223A2 (fr) * 2001-09-20 2003-03-27 The University Of Bath Ameliorations apportees au criblage d'insecticides ou s'y rapportant

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DABORN P ET AL: "DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid" MGG MOLECULAR GENETICS AND GENOMICS, vol. 266, no. 4, December 2001 (2001-12), pages 556-563, XP002277771 & ISSN: 1617-4615 *
DUNKOV B C ET AL: "THE DROSOPHILA CYTOCHROME P450 GENE CYP6A2: STRUCTURE, LOCALIZATION, HETEROLOGOUS EXPRESSION, AND INDUCTION BY PHENOBARBITAL" DNA, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 16, no. 11, November 1997 (1997-11), pages 1345-1356, XP008020802 ISSN: 1044-5498 *
TIJET NATHALIE ET AL: "The cytochrome P450 gene superfamily in Drosophila melanogaster: Annotation, intron-exon organization and phylogeny" GENE (AMSTERDAM), vol. 262, no. 1-2, 10 January 2001 (2001-01-10), pages 189-198, XP002277772 & ISSN: 0378-1119 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008083598A1 (fr) * 2006-12-30 2008-07-17 Zhicheng Shen Culture transgénique sélectivement éliminée, préparation et utilisation correspondantes
CN104059931A (zh) * 2014-07-03 2014-09-24 西南大学柑桔研究所 桔小实蝇细胞色素p450基因的rna干扰载体及其构建方法和应用
CN109112117A (zh) * 2017-09-04 2019-01-01 华中农业大学 一种分离的二化螟cyp15c1基因及其编码蛋白

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