[go: up one dir, main page]

WO2004044150A2 - Systemes permettant de cibler des genes et de realiser des insertions transgeniques genomiques stables - Google Patents

Systemes permettant de cibler des genes et de realiser des insertions transgeniques genomiques stables Download PDF

Info

Publication number
WO2004044150A2
WO2004044150A2 PCT/US2003/035587 US0335587W WO2004044150A2 WO 2004044150 A2 WO2004044150 A2 WO 2004044150A2 US 0335587 W US0335587 W US 0335587W WO 2004044150 A2 WO2004044150 A2 WO 2004044150A2
Authority
WO
WIPO (PCT)
Prior art keywords
transposon
half side
cassette
dna
site
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2003/035587
Other languages
English (en)
Other versions
WO2004044150A3 (fr
Inventor
Alfred M. Handler
Carsten Horn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Agriculture USDA
Original Assignee
US Department of Agriculture USDA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Agriculture USDA filed Critical US Department of Agriculture USDA
Priority to US10/534,226 priority Critical patent/US7700356B2/en
Priority to AU2003290660A priority patent/AU2003290660A1/en
Publication of WO2004044150A2 publication Critical patent/WO2004044150A2/fr
Publication of WO2004044150A3 publication Critical patent/WO2004044150A3/fr
Anticipated expiration legal-status Critical
Priority to US12/218,142 priority patent/US20090083870A1/en
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/65—Genetically modified arthropods
    • A01K67/68—Genetically modified insects
    • 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
    • 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)
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • both systems make possible the physical deletion of mobile DNA-sequences, brought in with the vector, from the target genome and therefore to stabilize the gene-of-interest.
  • Stable (genomic) transgene insertions are regarded to be an essential pre-requisite for the safe production of genetically modified organisms at a large industrial scale.
  • TransposonL The two ends of such a transposable element carrying within all functional parts necessary and sufficient for in vivo mobilization are termed TransposonL (5' end) and TransposonR (3' end).
  • TransposonL The transformation process is catalyzed by the transposase enzyme provided by a helper plasmid. This enzyme recognizes DNA target sites flanking the gene-of-interest/transgene and mobilizes the transgene into the genome of germ-line cells of the insect species.
  • transformed DNA contains a marker gene that allows detection of successful germ-line transformation events (by producing a dominantly visible phenotype).
  • EP 0 955 364 A36, the contents of which are incorporated herein by reference) and mariner are currently state- of-the-art technology to genetically modify important pest or useful insect species including, for example, malaria transmitting anopheline or culicine mosquitoes (Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Aedes aegypti; see Catteruccia, F., Nolan, T., Loukeris, T.G., Blass, C, Savakis, C, Kafatos, F.C.
  • malaria transmitting anopheline or culicine mosquitoes Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Aedes aegypti; see Catteruccia, F., Nolan, T., Loukeris
  • the application potential of broad host range transposable elements is not restricted to insect species: warmer-derived transformation vectors have been shown to integrate stably into the germ-line of the nematode, Caenorhabditis elegans (see Bessereau, J.-L., Wright, A., Williams, D.C., Schuske, K., Davis, M.W. & Jorgensen, E.M. (2001). Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 70-74, the contents of which are incorporated herein by reference), the zebrafish, Danio rerio (see Fadool J.M., Haiti, D.L.
  • Species-independent markers consist of a combination of a promoter sequence which is phylogenetically conserved and a gene for a fluorescent protein placed under control of such a promoter (for example, GFP [green fluorescing protein] and derivatives thereof, or DsRed [Discosoma species red fluorescing protein] (see Chalfie, M.
  • the polyubiquitin-promoter (see Patent Cooperation Treaty PCT WO 01/14537 Al and Handler, A.M. & Harrell, R.A. (1999). Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Mol. Biol. 8, 449-457, the contents of which are incorporated herein by reference) as well as the "3xP3"-promoter (see Patent Cooperation Treaty PCT WO 01/12667 Al and Berghammer, A.J., Klingler, M., & Wimmer, E.A. (1999). A universal marker for transgenic insects. Nature 402, 370-371 , the contents of which are incorporated herein by reference) linked to genes for fluorescent proteins have been used most widely for this purpose.
  • a transposon-independent technology aiming at targeting a gene-of-interest/transgene into the genome of cells relies on the principle of site-specific recombination. This is possible by using a recombinase enzyme and corresponding DNA target sites that are heterospecific.
  • the steps are: First, incorporating into the genome by transposon-mediated transformation, a DNA cassette that is flanked by heterospecific recombinase target sites and contains a marker system for positive-negative selection. Second, recombinase-mediated targeting into the marked genomic locus the gene-of-interest, which is located within a plasmid and is flanked by the same heterospecific recombinase target sites.
  • DNA cassette exchange systems has been demonstrated in different cell lines (comprising also murine embryonic stem cells) using the FLP-recombinase enzyme and heterospecific FRT target sites (see Schlake, T. & Bode, J. (1994). Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33, 12746-12751, and Seibler, J., Sch ⁇ beler, D., Fiering, S, Groudine, M. & Bode, J. (1998). DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker- free constructs.
  • Transposon-based plasmid vectors have proven to be efficient tools for producing genetically modified insects for research purposes, but so far only on a small laboratory scale.
  • the mobile nature of DNA transposable elements will be disadvantageous when scaling up the production/rearing of genetically modified insects. Owing to potential re-mobilization, the stability of genomic transgene integrations cannot be assured and, connected to this issue, concerns relating to the safety of release of such genetically modified insects will be raised. Stability of genomic transgene integrations in large industrial scale
  • the current state-of-the-art provides, typically, for random transposon vector integrations into the host genome. While this may be advantageous for functional genomics studies that use vector integrations to cause random mutations (e.g. for transposon-tagging and enhancer trapping), it is typically disadvantageous for the creation of transgenic strains for applied use where high fitness levels and optimal transgene expression are desired. This results from integrations that create mutations by insertion into genomic sites that eliminate or disrupt normal gene function that negatively effect viability, reproduction, or behavior. Genomic position effects also influence expression of transgenes, typically causing decreased expression and or mis-expression of genes of interest and markers so that transformants may not be easily identified, and the desired transgene expression for application is not achieved.
  • transposase enzyme corresponding to, and acting upon, the transposon sequences flanking the genomic transgene.
  • transposase enzyme corresponding to, and acting upon, the transposon sequences flanking the genomic transgene.
  • transposase used for germ-line transformation usually is not encoded by the host species' genome, transposase introduction by symbiotic or infectious agents is possible, and cross-reactivity to related transposase enzymes that are genomically encoded cannot be excluded.
  • Genome 37, 72-82 the contents of which are incorporated herein by reference).
  • translocation strains constructed for the Mediterranean fruit fly (see Franz, G., Gencheva, E. & Kerremans, Ph. (1994). Improved stability of genetic sex-separation strains for the Mediterranean fruit fly, Ceratitis capitata.
  • Genome 37, 72-82 the contents of which are incorporated herein by reference) suffered from instability. Recombination events causing reversion of the selected recessive trait were observed at a frequency of 10 "3 - 10 " (see Franz, G. (2002).
  • Biosafety includes minimizing the risk of unintended transgene transmission from the host to other procaryotic or eucaryotic species during rearing or after release into the field. Horizontal gene transfer cannot be excluded per se, because the mechanisms of nucleic acid exchange between species are not sufficiently investigated to date.
  • transposon vectors While most transposon vectors have their transposase source eliminated and are not self-mobilizable, functional autonomous transposons can be transmitted among species horizontally, and transposase may be provided to the vector by associated organisms or by a related enzyme in the host species.
  • the risk for transgene vector re-mobilization by a transposase-mediated event can be most definitively eliminated when transposon sequences, required for germ-line transformation, are removed from the genomic integration after the transformation process.
  • Systems disclosed in this patent application contribute to risk minimization by introducing techniques for transposon sequence removal.
  • Marker genes that can be distinguished from one another are placed in-between TransposonLl and TransposonL2 and in-between TransposonL2 and TransposonRl.
  • the steps of transformation are as follows. First, the transformation procedure is carried out according to the current state-of-the-art germline transformation technology that will result in individuals transformed by one of two possible events with this vector. One possible event is the integration of TransposonLl and TransposonRl and all intervening DNA including the two marker genes, TransposonL2, and other genes of interest. The second possible event is integration of TransposonL2 and TransposonRl and all intervening DNA including the marker gene.
  • the remaining TransposonLl half-side, with the downstream marker gene and genes-of- interest, is identified by the single marker gene phenotype and verified by sequencing of amplified DNA.
  • This remaining TransposonLl half side, marker gene and genes-of-interest should be incapable of re-mobilization by transposase in the absence of the requisite TransposonRl half side.
  • the second embodiment disclosed has been termed "conditional excision-competent transformation vectors" (Fig. 4).
  • This embodiment comprises a modified excision-competent transformation vector that contains a transposonR2 half-side in an inverted orientation, relative to the Rl half side, with R2 also flanked by recombinase target sites in inverted orientation.
  • R2 also flanked by recombinase target sites in inverted orientation.
  • only the TransposonLl and Rl half-sides can integrate by transposition, and remobilization of the TransposonLl and R2 half-sides can only occur after a recombinase- mediated inversion between the recombinase target sites.
  • RMCE with subsequent transposon deletion (Fig. 5).
  • Completely new in this embodiment is a DNA targeting strategy.
  • the ultimate germ-line transformation process is conducted as a recombinase-mediated process, instead of a transposase-mediated process, into an existing (and pre-defined) genomic target site.
  • This involves the RMCE principle i.e. a site-specific recombinase recognizes heterospecific DNA target sites and exchanges DNA-cassettes between a RMCE-acceptor and a RMCE-donor (step 1 in Fig. 5).
  • the success of this cassette exchange is indicated by the exchange of the acceptor target marker gene (e.g. ECFP, see Fig.
  • the donor vector marker gene e.g. EYFP, see Fig. 7
  • EYFP the donor vector marker gene
  • the advantage of this promoter-free exchange is that side-reactions, which involve non-targeted integration of the donor into the genome, will not be recognized.
  • Most important to this first step of cassette exchange is a "homing DNA sequence" that is present in both the RMCE-acceptor and the RMCE-donor and is identical in both functional parts. The homing DNA sequence functions to significantly enhance the cassette exchange efficiency.
  • Fig. 1 shows a protocol for integration and re-mobilization for stabilized vector creation
  • Fig. 2 shows a diagram of stabilization vector pBac (LI -PUbDsRedl-L2-3xP3-ECFP-Rl ⁇
  • Fig. 3 shows a PCR analysis and verification of pBac ⁇ Ll-PUbDsRedl-L2-3xP3-ECFP-Rl ⁇ vector integration in line F34 and L2-3xP3-ECFP-Rl remobilization in line F34-1M
  • Fig. 4 shows the principle of "conditional excision competent transformation vectors"
  • Fig. 5 shows the principle of "RMCE with subsequent transposon deletion”
  • Fig. 6 shows an embodiment of the principle as shown in Fig. 4
  • Fig. 8 shows a diagram of RMCE acceptor vector
  • Fig. 9 shows molecular analysis of RMCE acceptor and RMCE donor transgenic lines
  • Fig. 10 shows a diagram of a final RMCE donor vector for transgene stabilization
  • Fig. 11 shows the approximate sequence of the vector shown in Fig. 2;
  • Fig. 12 shows the approximate sequence of the vector shown in Fig. 8.
  • transposition can occur utilizing the LI and Rl half sides, or the internal L2 and Rl half sides.
  • pBac ⁇ Ll-PUbDsRedl-L2-3xP3-ECFP-Rl ⁇ contains a unique Kasl restriction endonuclease site in the piggyBacT ⁇ region that can be used to insert genes of interest.
  • independent transformation marker genes are placed in-between the two half-side pairs.
  • Genomic insertion site DNA flanking the integration was obtained by inverse PCR of the piggyBac l 5'-end half side using the 122R and 139F primers, in outward orientation, to F34 genomic DNA digested with Mspl endonuclease and circularized by ligation.
  • the 5 'end insertion site sequence was compared by BLAST analysis to the Drosophila Genome Sequence Database, and consistent with segregation analysis, was found to be homologous to sequence found on the X-chromosome at locus 9B4.
  • the internal transposon half-side (R2) is a duplication of the piggyBac 3 '-end, and it is in reverse, or opposite, orientation to Rl.
  • R2 the internal transposon half-side
  • FRT FLP recombinase target
  • pBac STBL contains unique cloning sites for the rare octamer-specific restriction enzymes Ascl and Fsel.
  • pBac_STBL is equipped with two separable transformation marker genes (see WO 01/12667, the contents of which are incorporated herein by reference), which are located upstream of the Ascl/Fsel cloning sites (3xP3-EYFP; Fig. 6) and downstream of the FRT-sites (3xP3-DsRed; Fig. 6), respectively.
  • P SL-3xP3-DsRedaf the details of pBac STBL plasmid construction starting from plasmid vectors already published are disclosed:
  • Genomic integrations of the pBac_STBL transgene are identifiable by both EYFP and DsRed eye fluorescence (see WO 01/12667, the contents of which are incorporated herein by reference).
  • the inversion of the piggyBacRl sequence is carried out. This is performed by crossing in the strain beta2t-E P that expresses FLP-recombinase during spermatogenesis. Alternatives of step 2 in Fig.
  • ECFP progeny (selection against the jumpstarter) of single male crossings are analyzed for both the presence of EYFP fluorescence and the absence of DsRed fluorescence. Individuals putatively containing a transposon deletion event should show EYFP but absence of DsRed fluorescence and can be analyzed further.
  • the transposon deletion can be molecularly confirmed and stability of the potentially immobilized transgene insertion can be assessed by challenging the transgene insertion with piggyBac transposase.
  • Embodiment 3 RMCE with subsequent transposon deletion
  • the RMCE-acceptor plasmid, pBac ⁇ 3xP3-FRT-ECFP-linotte-FRT3 ⁇ (Fig. 8), is & piggyBac- based transformation vector that was provided additionally with a DNA exchange cassette.
  • This cassette consists of two heterospecific FRT sites (refe ⁇ ed to as FRT and FRT3 equivalent to F and F3 (published in European Patent No. EP 0 939 120 Al, the contents of which are incorporated herein by reference)) in parallel orientation.
  • EP 0 939 120 Al (see page 2, line 50 to page 3, line 6) teaches the technology of the RMCE reaction:
  • 3xP3-FRT- ⁇ CFPaf was cloned into the plasmid p3E1.2 previously digested with Hpal.
  • the plasmid pBac ⁇ 3xP3-FRT-ECFPaf ⁇ was digested with Ascl-BgHl, and the following sequences were cloned into the linearized vector: i.) the Ascl-Aspl IS cut PCR amplification product of the 1.6 kb H dIII genomic linotte fragment.
  • genomic D ⁇ A of Drosophila melanogaster, strain OregonR was chosen and as primers:
  • C ⁇ _lioRev (5'-CGGGGTACCCCAAGCTTATTAGAGTAGTATTCTTC-3') and ii.) the AspllS-Bg ⁇ Tl cut PCR amplifcation product of the FRT3 sequence (mutagenic PCR).
  • the plasmid pSL>AB> was chosen and as primers:
  • the 3xP3 promoter sequence was deleted from the plasmid pSL-3xP3-FRT-EYFPaf by digestion with EcoRI-R ⁇ wHI, filling-in by Klenow enzyme reaction and finally religating the blunted plasmid.
  • pSL-FRT-EYFP-linotte-FRT3 The 3xP3 promoter sequence was deleted from the plasmid pSL-3xP3-FRT-EYFPaf by digestion with EcoRI-R ⁇ wHI, filling-in by Klenow enzyme reaction and finally religating the blunted plasmid.
  • pSL-FRT-EYFP-linotte-FRT3 was deleted from the plasmid pSL-3xP3-FRT-EYFPaf by digestion with EcoRI-R ⁇ wHI, filling-in by Klenow enzyme reaction and finally religating the blunted plasmid.
  • pSL-FRT-EYFP-linotte-FRT3
  • FRT-ECFP-linotte-FRT3 ⁇ was cloned into the plasmid pSL-FRT-EYFPaf previously digested with Nrul. The orientation with maximal distance of the FRT and FRT3 sites was chosen.
  • pBac ⁇ 3xP3-DsRedafl
  • DsRedaf was cloned into the plasmid pSL-FRT- ⁇ YFP-linotte-FRT3 previously digested with
  • DsRedaf ⁇ was cloned into the plasmid pSL-FRT- ⁇ YFP-linotte-FRT3 previously cut with
  • FLP recombinase plasmid source p ⁇ ihsp82-FLP:
  • helper phspBac was used (see PCT WO
  • RMCE donor plasmid pSL-FRT-EYFP-linotte-FRT3 and the FLP recombinase expression vector pKhsp82-FLP were co-injected into pre-blastoderm embryos of a Drosophila melanogaster acceptor strain. These embryos cany the RMCE acceptor transgene vector (Fig. 8) integrated by piggyB ac-mediated germ-line transformation, in a homozygous state.
  • the final concentration of the plasmids in the injection mix was 500 ng/ ⁇ l (RMCE donor plasmid) and 300 ng/ ⁇ l (pKhsp82-FLP).
  • Table 1 Results of the RMCE experiment in Drosophila with the donor plasmid pSL-FRT- EYFP-linotte-FRT3.
  • Acceptor lines II: second, III: third chromosomal homozygous, ECFP fluorescence
  • EYFP-positive founder males resulting from targeting events were bred to homozygosity and established as stocks (refe ⁇ ed to as "M4.II EYFP", "M7.III EYFP”, “M8.II EYFP” and "M9.II EYFP", respectively).
  • Segregation analysis (genetic mapping of transgene integrations) indicated for all four lines that the chromosomal localization of the donor and acceptor transgene is identical.
  • the DNA cassette exchange frequency is a percentage of fertile Fj vials producing EYFP-positive progeny.
  • the frequency of RMCE events is 25% on average co ⁇ esponding well to the germ-line transformation frequency usually observed with piggyBac, Hermes or os-based vectors mDrosophila).
  • This experiment demonstrates that, with the particular design of RMCE-vectors, the process of cassette exchange is highly efficient in an invertebrate organism such as Drosophila.
  • Molecular characterization of RMCE events and integration site analysis a) Genomic integration site of donor and acceptor transgenes
  • genomic integration sites of the acceptor transgene in the acceptor line and of the donor transgene in the co ⁇ esponding donor line should be identical.
  • inverse PCR experiments were carried out for acceptor and donor Drosophila lines.
  • inverse PCR was performed. The purified fragments were directly sequenced for the 5' junction with primer CH_PLSeq 5'-CGGCGACTGAGATGTCC-3'. The obtained sequences were used in BLAST searches against the Drosophila Genome Sequence Database. For the 5' junction, genomic DNA sequence identity could be confirmed for three acceptor/donor pairs (Table 2).
  • acceptor line M9.II ECFP was found to cany the acceptor transgene integrated at the Drosop ⁇ -endogenous linotte locus (integration position co ⁇ esponds to bp
  • the expected pattern of DNA-DNA hybridization 2.4 kb for the acceptor transgene and 1.6 kb for the donor transgene, was detected for all four lines for each transgene (Fig. 9). Additionally, a ⁇ 6 kb hybridization signal was detected only in RMCE donor lines. As this signal might indicate the presence of the complete donor vector, further Southern experiments (using probes against the pUC plasmid backbone sequences) were carried out. The presence of pUC sequence in the donor lines could be confirmed (data not shown) pointing toward an integration of the entire donor vector in the four donor lines analyzed.
  • the recombinase-mediated cassette exchange mechanism requires a double recombination event (see European Patent No. EP 0 939 120, the contents of which are incorporated herein by reference). Because the Southern analysis suggests that in the pilot RMCE experiments single recombination events caused integration of the entire donor plasmid, we analyzed in more detail whether the RMCE mechanism, which has not been established for an invertebrate organism, can occur in Drosophila. To this end, we modified the donor construct to include a 3xP3-DsRed marker gene downstream to the FRT3 sequence (pSL-FRT-EYFP-linotte-FRT3- 3xP3-DsRed). This vector configuration allows the separation of RMCE events: 1) double cross-over via FRT and FRT3 sites resulting in ECFP to EYFP eye fluorescence exchange
  • the acceptor line M4.II ECFP (Tablel) was selected for further testing.
  • FI individuals with ECFP to EYFP exchange indicating targeting were observed at a frequency of 13.1 %:
  • Table 3 Phenotypic analysis of FI progeny from GO male founders of the acceptor line M4.II ECFP injected with the donor pSL-FRT-EYFP-linotte-FRT3-3xP3-DsRed. Double and single recombination events are indicated by differential analysis of eye fluorescence for ECFP, EYFP and DsRed.
  • RMCE strategy can be further employed for the purpose of post-transformational transgene immobilization.
  • the general procedure consists of two steps. In the first step, a transformation vector containing the gene of interest, a transposon half-side (TransposonR2 in Fig. 5) and an additional marker gene is used as the RMCE donor to target the RMCE acceptor line (i.e. RMCE acceptor vector (Fig.8) genomically integrated).
  • an 'internal' piggyBac transposon comprising both half-sides (piggyBacLl and piggyBacR2 in Fig. 5) is reconstituted.
  • transposase activity is introduced to remobilize the 'internal' transposon by selecting for individuals lacking the additional marker gene as demonstrated in embodiment 1.
  • Stepl Targeted DNA cassette exchange (RMCE. Step 1 in Fig. 5 and Fig. 7)
  • final RMCE donor contains, in-between the FRT and FRT3 sites, a cassette with: (i) a promotor-free eyfp ORF, (ii) the piggyBacRl (3' end) transposon sequence, (iii) the transformation marker 3xP3-DsRed, and (iv) the homing sequence from the Drosophila linotte locus (see Taillelaub, E. & Dura, J.M. (1999). A novel mechanism for P element homing in Drosophila. Proc. Natl. Acad. Sci. USA 96, 6856-6861, the contents of which are incorporated herein by reference).
  • Derivatives of the final RMCE donor vector carrying additional DNA sequences can be constructed by insertion into the unique Ascl and Fsel cloning sites which are located upstream of the piggyBacRl transposon sequence (Fig. 10).
  • Progeny (generation F2) carrying both the final RMCE donor and the jumpstarter transgenes were crossed individually to non-transgenic Drosophila and progeny from these crosses (generation F3) were analyzed for the presence of individuals carrying EYFP but lacking DsRed eye fluorescence (Table 4). Js HerM6 HerMlO MiM5
  • the particular embodiments of the invention are highly flexible.
  • the functionality of systems disclosed is neither dependent on the particular transposable elements used in the embodiments, nor on the particular transformation marker genes used in the embodiments, nor on the particular site-specific recombination system used in the embodiments, nor on the particular homing sequence used in embodiment 3.
  • all embodiments have broad general application potential in vertebrate and invertebrate organisms that are subject to transposon-mediated transformation or recombinase-mediated recombination, and fluorescent protein marking systems.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Environmental Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne de nouveaux systèmes de transformation de cellules germinales permettent de supprimer physiquement l'ADN du transposon après le processus de transformation et de cibler des intégrations transgéniques dans des sites cibles prédéfinis. De cette manière, la mobilisation des gènes d'intérêt médiée par la transposase est exclue de façon mécanistique et les intégrations génomiques aléatoires sont éliminées. Contrairement aux techniques classiques de transformation de cellules germinales, les systèmes selon l'invention renforcent la stabilité de l'insertion transgénique. De plus, les séquences ADN requises pour la modification transgénique (par exemple, des gènes marqueurs de transformation, des sites cibles de transposase ou de recombinase) sont largement supprimées du génome après l'insertion transgénique finale, éliminant ainsi la possibilité d'une instabilité générée par ces processus. La technologie RMCE, qui est décrite dans cette demande de brevet pour des organismes invertébrés (par exemple, Drosophila melanogaster) représente un outil extrêmement polyvalent ayant un potentiel d'application qui dépasse largement le but de l'immobilisation transgénique. La technologie RMCE rend possible l'intégration ciblée de cassettes d'ADN dans des loci génomiques spécifiques qui sont prédéfinis par l'intégration du plasmide de l'accepteur RMCE. Ces loci peuvent être caractérisés avant une expérience de ciblage de façon que les sites d'intégration optimaux puissent être présélectionnés pour des applications spécifiques et que des souches hôtes présentant une santé optimale puissent être sélectionnées. De plus, plusieurs réactions d'échange de cassettes peuvent être réalisées de façon répétitive de sorte qu'une cassette d'accepteurs peut être échangée de façon répétitive par plusieurs cassettes de donneurs. Ainsi, différents transgènes peuvent être placés avec précision sur le même locus génomique, ce qui permet, pour la première fois, d'éliminer les effets positionnels génomiques et d'étudier de façon comparative les effets biologiques de différents transgènes.
PCT/US2003/035587 2002-11-07 2003-11-07 Systemes permettant de cibler des genes et de realiser des insertions transgeniques genomiques stables Ceased WO2004044150A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/534,226 US7700356B2 (en) 2002-11-08 2003-11-07 System for gene targeting and producing stable genomic transgene insertions
AU2003290660A AU2003290660A1 (en) 2002-11-07 2003-11-07 Systems for gene targeting and producing stable genomic transgen e insertions
US12/218,142 US20090083870A1 (en) 2002-11-07 2008-07-11 Systems for gene targeting and producing stable genomic transgene insertions

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10251918.8 2002-11-07
DE10251918A DE10251918A1 (de) 2002-11-08 2002-11-08 Systeme zur Erzeugung stabiler genomischer Transgen-Insertionen

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/218,142 Division US20090083870A1 (en) 2002-11-07 2008-07-11 Systems for gene targeting and producing stable genomic transgene insertions

Publications (2)

Publication Number Publication Date
WO2004044150A2 true WO2004044150A2 (fr) 2004-05-27
WO2004044150A3 WO2004044150A3 (fr) 2004-07-08

Family

ID=32115360

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/035587 Ceased WO2004044150A2 (fr) 2002-11-07 2003-11-07 Systemes permettant de cibler des genes et de realiser des insertions transgeniques genomiques stables

Country Status (3)

Country Link
AU (1) AU2003290660A1 (fr)
DE (1) DE10251918A1 (fr)
WO (1) WO2004044150A2 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7998475B2 (en) * 2003-05-12 2011-08-16 Oxitec Limited Dilution of genetic traits
US8124404B2 (en) 2003-07-01 2012-02-28 Oxitec Limited Stable integrands
US9121036B2 (en) 2003-07-28 2015-09-01 Oxitec Limited Expression system for insect pest control
US9125388B2 (en) 1999-11-29 2015-09-08 Isis Innovation Limited Biological control
US9487801B2 (en) 2012-03-05 2016-11-08 Oxitec Limited Biocontrol
US9504236B2 (en) 2009-07-08 2016-11-29 Kymab Limited Animal models and therapeutic molecules
US9788534B2 (en) 2013-03-18 2017-10-17 Kymab Limited Animal models and therapeutic molecules
US9970025B2 (en) 2006-02-10 2018-05-15 Oxitec Limited Gene expression system using alternative splicing in insects
WO2019037099A1 (fr) * 2017-08-25 2019-02-28 Wenning Qin Modification à grande échelle de génome eucaryote
US10426147B2 (en) 2013-03-05 2019-10-01 Oxitec Limited Insect muscle actin promoter
US10667501B2 (en) 2012-05-17 2020-06-02 Kymab Limited Transgenic non-human vertebrate for the in vivo production of dual specificity immunoglobulins or hypermutated heavy chain only immunoglobulins
US11051497B2 (en) 2011-09-19 2021-07-06 Kymab Limited Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics
US11297811B2 (en) 2012-03-28 2022-04-12 Kymab Limited Transgenic non-human vertebrate for the expression of class-switched, fully human, antibodies
US11399522B2 (en) 2013-10-01 2022-08-02 Kymab Limited Animal models and therapeutic molecules
US11564380B2 (en) 2009-07-08 2023-01-31 Kymab Limited Animal models and therapeutic molecules
US11707056B2 (en) 2013-05-02 2023-07-25 Kymab Limited Animals, repertoires and methods
US11737436B2 (en) 2014-06-05 2023-08-29 Oxitec Limited Gene expression system
US11820810B2 (en) 2013-05-02 2023-11-21 Kymab Limited Antibodies, variable domains and chains tailored for human use
US11827876B2 (en) 2016-08-12 2023-11-28 Oxitec Ltd. Self-limiting, sex-specific gene and methods of using
US12492404B2 (en) 2018-03-29 2025-12-09 Oxitec Limited Self-limiting noctuids

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2761008A1 (fr) 2011-09-26 2014-08-06 Kymab Limited Chaînes légères substituts (cls) chimères comprenant vpreb humain
US9253965B2 (en) 2012-03-28 2016-02-09 Kymab Limited Animal models and therapeutic molecules
US9783618B2 (en) 2013-05-01 2017-10-10 Kymab Limited Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0688358A4 (fr) * 1993-03-12 1997-10-01 Univ Creighton Vecteurs ameliores en therapie genique
CA2401677A1 (fr) * 2000-03-03 2001-09-13 University Of Utah Research Foundation Procede de ciblage genique
US20020094575A1 (en) * 2000-09-14 2002-07-18 Pioneer Hi-Bred International, Inc. Compositions and methods for stable transformation using Mu bacteriophage cleaved donor complex

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9125388B2 (en) 1999-11-29 2015-09-08 Isis Innovation Limited Biological control
US7998475B2 (en) * 2003-05-12 2011-08-16 Oxitec Limited Dilution of genetic traits
US8124404B2 (en) 2003-07-01 2012-02-28 Oxitec Limited Stable integrands
US9133477B2 (en) 2003-07-28 2015-09-15 Oxitec Limited Expression systems
US10059961B2 (en) 2003-07-28 2018-08-28 Oxitec Limited Expression systems
US9121036B2 (en) 2003-07-28 2015-09-01 Oxitec Limited Expression system for insect pest control
US10844402B2 (en) 2003-07-28 2020-11-24 Oxitec Limited Expression systems
US9970025B2 (en) 2006-02-10 2018-05-15 Oxitec Limited Gene expression system using alternative splicing in insects
US10941416B2 (en) 2006-02-10 2021-03-09 Oxitec Limited Gene expression system using alternative splicing in insects
US9504236B2 (en) 2009-07-08 2016-11-29 Kymab Limited Animal models and therapeutic molecules
US11812731B2 (en) 2009-07-08 2023-11-14 Kymab Ltd. Animal models and therapeutic molecules
US11606941B2 (en) 2009-07-08 2023-03-21 Kymab Limited Animal models and therapeutic molecules
US11564380B2 (en) 2009-07-08 2023-01-31 Kymab Limited Animal models and therapeutic molecules
US11051497B2 (en) 2011-09-19 2021-07-06 Kymab Limited Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics
US9487801B2 (en) 2012-03-05 2016-11-08 Oxitec Limited Biocontrol
US11297811B2 (en) 2012-03-28 2022-04-12 Kymab Limited Transgenic non-human vertebrate for the expression of class-switched, fully human, antibodies
US10667501B2 (en) 2012-05-17 2020-06-02 Kymab Limited Transgenic non-human vertebrate for the in vivo production of dual specificity immunoglobulins or hypermutated heavy chain only immunoglobulins
US10426147B2 (en) 2013-03-05 2019-10-01 Oxitec Limited Insect muscle actin promoter
US11297810B2 (en) 2013-03-18 2022-04-12 Kymab Limited Animal models and therapeutic molecules
US9788534B2 (en) 2013-03-18 2017-10-17 Kymab Limited Animal models and therapeutic molecules
US11707056B2 (en) 2013-05-02 2023-07-25 Kymab Limited Animals, repertoires and methods
US11820810B2 (en) 2013-05-02 2023-11-21 Kymab Limited Antibodies, variable domains and chains tailored for human use
US11399522B2 (en) 2013-10-01 2022-08-02 Kymab Limited Animal models and therapeutic molecules
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
WO2019037099A1 (fr) * 2017-08-25 2019-02-28 Wenning Qin Modification à grande échelle de génome eucaryote
US12492404B2 (en) 2018-03-29 2025-12-09 Oxitec Limited Self-limiting noctuids

Also Published As

Publication number Publication date
AU2003290660A8 (en) 2004-06-03
WO2004044150A3 (fr) 2004-07-08
AU2003290660A1 (en) 2004-06-03
DE10251918A1 (de) 2004-05-19

Similar Documents

Publication Publication Date Title
US20090083870A1 (en) Systems for gene targeting and producing stable genomic transgene insertions
WO2004044150A2 (fr) Systemes permettant de cibler des genes et de realiser des insertions transgeniques genomiques stables
Dupuy et al. Transposition and gene disruption in the male germline of the mouse
Vidal et al. Cre expression in primary spermatocytes: a tool for genetic engineering of the germ line
CN101297031B (zh) 在脊椎动物中作为遗传操作和分析工具的piggyBac
Auerbach Production of functional transgenic mice by DNA pronuclear microinjection.
Handler et al. Prospects for gene transformation in insects
EP1187938B1 (fr) Procede d'expression de transgenes dans la lignee germinale de caenorhabditis elegans
CA3004497A1 (fr) Insertion ciblee de sequence d'adn genomique volumineux et utilisations associees
WO1993004169A1 (fr) Ciblage de genes dans des cellules animales au moyen de produits de synthese d'adn isogeniques
CN104673815B (zh) 复合型piggyBac重组载体及其制备方法和应用
Geurts et al. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon
Takasu et al. Precise genome editing in the silkworm Bombyx mori using TALENs and ds-and ssDNA donors–A practical approach
Long et al. In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange
Rülicke et al. Germ line transformation of mammals by pronuclear microinjection
Amsterdam Insertional mutagenesis in zebrafish
Kaneko et al. Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection
EP1268837B1 (fr) PROCEDE DE PRODUCTION de MAMMIFERES TRANSGENIQUES A L'AIDE DE TRANSPOSONS
WO2002013602A1 (fr) Mammifere transgenique non humain et procede de realisation de ce dernier, modele de maladie animale et procede de clarification de la fonction genique
WO2001000809A1 (fr) Deletion d'adn auto-induite
Strojek et al. The use of transgenic animal techniques for livestock improvement
Enver et al. Analysis of the developmental and transcriptional potentiation functions of 5′ HS2 of the murine β-globin locus control region in transgenic mice
MacDonald et al. Analysis of transcriptional regulatory regions in vivo
US20200352143A1 (en) Method to Implement a CRISPR Gene Drive in Mammals
US20210251203A1 (en) Polynucleotide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2006218652

Country of ref document: US

Ref document number: 10534226

Country of ref document: US

122 Ep: pct application non-entry in european phase
WWP Wipo information: published in national office

Ref document number: 10534226

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: JP