US20170188559A1 - Gene expression system - Google Patents

Gene expression system Download PDF

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US20170188559A1
US20170188559A1 US15/313,922 US201515313922A US2017188559A1 US 20170188559 A1 US20170188559 A1 US 20170188559A1 US 201515313922 A US201515313922 A US 201515313922A US 2017188559 A1 US2017188559 A1 US 2017188559A1
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gene
polynucleotide sequence
seq
promoter
gene expression
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Martha KOUKIDOU
Luke Alphey
Simon Warner
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Oxitec Ltd
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Oxitec Ltd
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Assigned to OXITEC, LTD. reassignment OXITEC, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WARNER, SIMON, KOUKIDOU, Martha, ALPHEY, LUKE
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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
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    • 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
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • 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
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/203—Animal model comprising inducible/conditional expression system, e.g. hormones, tet
    • 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
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • 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
    • A01K2217/30—Animal model comprising expression system for selective cell killing, e.g. toxins, enzyme dependent prodrug therapy using ganciclovir
    • 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
    • A01K2267/02—Animal zootechnically ameliorated
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
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    • 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
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/007—Vectors comprising a special translation-regulating system cell or tissue specific
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/75—Vectors comprising a special translation-regulating system from invertebrates
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    • C12N2999/00—Further aspects of viruses or vectors not covered by groups C12N2710/00 - C12N2796/00 or C12N2800/00
    • C12N2999/007—Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Each of the two systems comprises a dominant lethal gene to be expressed and an activating transcription factor to activate expression of the lethal gene.
  • the effect of the activating transcription factor can be repressed, and the product of the dominant lethal gene has a lethal effect on the insect when expressed in sufficient quantity.
  • Each expression system also comprises a splice control sequence which provides for female-specificity of the lethal effect.
  • the presence of two female-specific, repressible, dominant, lethal expression systems improves the penetrance of the system by increasing the amount of lethal product expressed, thereby increasing the probability of effective lethality.
  • the presence of two expression systems also induces earlier onset of lethality during development due to an accumulation of lethal product, and the risk of resistance mechanisms is reduced because the probability of developing resistance to both expression systems is low.
  • SEQ ID NO: 6 shows a nucleotide sequence of primer TG2-3864FRTFIF.
  • SEQ ID NO: 21 shows a nucleotide sequence of primer Diag2-hr5.
  • SEQ ID NO: 27 shows a nucleotide sequence of the tTAV2 gene.
  • At least one of the promoters is a minimal promoter.
  • each of the promoters is independently Hsp70, Hsp73 or sry ⁇ .
  • one of the first and second promoters is Hsp70 and the other is sry ⁇ .
  • the first promoter is Hsp70 and the second promoter is sry ⁇ .
  • the first and second lethal genes comprise a coding sequence for a protein or polypeptide, i.e. at least one exon, and preferably two or more exons, capable of encoding a polypeptide, such as a protein or fragment thereof.
  • the different exons are differentially spliced together to provide alternative mRNAs.
  • said alternative spliced mRNAs have different coding potential, i.e. encode different proteins or polypeptide sequences.
  • the expression of the coding sequence is regulated by alternative splicing.
  • the manner or mechanism of alternative splicing is sex-specific, preferably female-specific, and any suitable splice control sequence may be used.
  • at least one splice control sequence is derived from a tra intron.
  • the Ceratitis capitata tra intron from the transformer gene was initially characterised by Pane et al (2002), supra.
  • the TRA protein is differentially expressed in different sexes.
  • the TRA protein is known to be present largely in females and, therefore, mediates alternative splicing in such a way that a coding sequence is expressed in a sex-specific manner, i.e.
  • At least one of the splice control sequences is derived from the alternative splicing mechanism of the Actin-4 gene derived from an arthropod, preferably a tephritid. In embodiments wherein more than one splice sequence is derived from Actin-4, they may be derived from the same or from different tephritid species. In some embodiments, each Actin-4 gene is independently derived from a species of the Ceratitis , the Bactrocera , the Anastrepha or the Rhagoletis genera.
  • both the first and the second gene expression system further comprise an enhancer, i.e. first and second enhancers, respectively.
  • one of the first and second enhancers is tetO ⁇ 7 and the other enhancer is tetO ⁇ 14.
  • the first enhancer is tetO ⁇ 7 and the second enhancer is tetO ⁇ 14.
  • the first and second gene expression systems are arranged in tandem, forming a transgene, and the transgene may or may not comprise linker sequences of nucleotides between each gene expression system.
  • the first and second gene expression systems are contiguous.
  • the linker sequence is from 1 bp to 10 kbp in length.
  • the construct comprises four piggyBac inverted repeats forming at least two pairs of opposing inverted repeats.
  • the four piggyBac inverted repeats consist of the nucleotide sequences represented by SEQ ID NOs: 30-32, with the sequence represented by SEQ ID NO: 30 being used for two of the piggyBac inverted repeats.
  • one external inverted repeat consists of the nucleotide sequence represented by SEQ ID NO: 30 and the other external inverted repeat consists of the nucleotide sequence represented by SEQ ID NO: 31.
  • embodiments comprising at least one genetic marker will also comprise a promoter to drive the expression of the genetic marker.
  • the promoter is Hr5/IE1 (Choi & Guarino, 1995), while in other embodiments the promoter is Polyubiquitin (Handler & Harrel, 2001).
  • promoters for use with the genetic marker are not limited to these two examples, and others may be used. Those skilled in the art will recognise which promoters are suitable.
  • the method for detecting an organism transformed with a transgene or construct as described above further comprises the use of a dual-labelled probe during the amplification steps.
  • strain OX3864E contained a silent insertion (confirmed by flanking sequence analysis) and was discarded. All remaining strains were positive for homozygosis potential and thus crossed to the medfly strain OX3133 for piggyBac excision (Dafa'alla et al., 2006). Only strains OX3864A and OX3647Q demonstrated complete removal of all piggyBac sequences and were thus selected as potential product strains.
  • strains OX3864A and OX3647Q were further tested for life history parameters in comparison to the TOLIMAN wild-type strain that they were both derived from and also the temperature-sensitive-lethal (tsl) Vienna 8 strain (reference).
  • tsl is the genetic sexing strain T(Y;5)101 called also Vienna ⁇ 8 (without the pericentric inversion D53) (Caceres, 2002), introgressed into the TOLIMAN wt that is currently used in many Sterile Insect Technique (SIT) programmes worldwide. Details of the tests performed are given below. Graphs are presented in FIG. 4 . Fitness indices are given in Table 2.
  • Genomic DNA was isolated from individual insects using the protocol below (also found in TD/SOP/00142) using the Invitrogen Purelink genomic extraction kit.

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Cited By (4)

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WO2020035673A1 (en) 2018-08-14 2020-02-20 Oxitec, Ltd. Self-selecting sterile male arthropods
US11737436B2 (en) 2014-06-05 2023-08-29 Oxitec Limited Gene expression system
US11827876B2 (en) 2016-08-12 2023-11-28 Oxitec Ltd. Self-limiting, sex-specific gene and methods of using
US11968963B2 (en) 2018-04-05 2024-04-30 Vanderbilt University Male arthropod killing factors and methods of use thereof

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GB2540786A (en) * 2015-07-28 2017-02-01 Glaxosmithkline Ip Dev Ltd Codon optimised tet repressor proteins
CN108728477B (zh) * 2017-04-24 2022-02-22 华东理工大学 一种高效的转座突变系统及构建方法

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