EP1006790A1 - UTILISATION DE L'ELEMENT $i(MARINER) POUR CREER DES ANIMAUX TRANSGENIQUES - Google Patents

UTILISATION DE L'ELEMENT $i(MARINER) POUR CREER DES ANIMAUX TRANSGENIQUES

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Publication number
EP1006790A1
EP1006790A1 EP98939777A EP98939777A EP1006790A1 EP 1006790 A1 EP1006790 A1 EP 1006790A1 EP 98939777 A EP98939777 A EP 98939777A EP 98939777 A EP98939777 A EP 98939777A EP 1006790 A1 EP1006790 A1 EP 1006790A1
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EP
European Patent Office
Prior art keywords
embryo
transposase
mariner
ammal
cell
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.)
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Application number
EP98939777A
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German (de)
English (en)
Inventor
Helen Roslin Institute SANG (Edinburgh)
David John University of Edinburgh FINNEGAN
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Roslin Institute Edinburgh
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Biotechnology and Biological Sciences Research Council
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Priority claimed from GBGB9717913.9A external-priority patent/GB9717913D0/en
Priority claimed from GBGB9812822.6A external-priority patent/GB9812822D0/en
Application filed by Biotechnology and Biological Sciences Research Council filed Critical Biotechnology and Biological Sciences Research Council
Publication of EP1006790A1 publication Critical patent/EP1006790A1/fr
Withdrawn legal-status Critical Current

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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
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, 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
    • 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
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a method for introducing a transgene into an animal embryo and to the preparation of a transgenic animal therefrom.
  • the frequency at which this chromosomal integration occurs varies between species.
  • the frequency is also influenced by the site of injection. If the DNA is introduced into one of the pronuclei of the fertilised egg, the frequency of production of transgenic animals is generally higher.
  • Transposable elements are defined sequences of DNA that can transpose to different sites in the genome of an organism. Transposable elements can be divided into several different classes, defined by the mechanism which they use to move from one genomic site to another. The ability of transposable elements has been modified to enable use of a transposable element from a particular species to be used as a vector to introduce foreign DNA into the genome of that species, e.g. the P element from Drosophila melanogaster is widely used to transform D. melanogaster (Rubin, G. M., & Spradling, A. C, Science 218 348-353 (1982) and US-A-4670388).
  • the efficiency of this method is therefore relatively poor with only 1% or less of chicks hatched after DNA injection have incorporated the injected DNA into their genome. Additionally, from each of these transgenic birds only a single transgenic line has been obtained, i.e. the transgenic offspring contain a few copies of the foreign DNA at a single chromosome site.
  • Retroviral vectors Transgenic birds have also been produced using infection of retroviral vectors (Bosselman et al Science 243 533-535 (1989)).
  • retroviral vectors have several disadvantages. The risk of recombination of viral vectors with wild type retroviruses which are widespread in poultry populations is perceived as the most serious problem. Retroviral vectors are also very complicated to work with and are restricted in their capacity to incorporate constructs greater than approximately 8 kilobases of DNA.
  • transposable element mariner was originally discovered in the genome of Drosophila mauritiana but closely related elements have been discovered in a wide variety of species both vertebrate and invertebrate (Robertson, H. M., Nature 362 241 (1993)) It has also been used to investigate pathogenic orgamsms such as Leishmama (Gueiros-Filho, F J., & Beverley, S.
  • the mariner element can be used as a vector in the preparation of transgenic animals
  • a method for the preparation of a transgenic animal embryo composing the step of introducing a mariner-like element (MLE) containing a transgene into an animal embryo cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encodmg a transposase
  • MLE mariner-like element
  • transgenic is used m the context of the present invention to desc ⁇ be animals which have stably incorporated a sequence of foreign DNA introduced by the mariner-like element (MLE) into their chromosomes such it that may passed on to successive generations of transgenic descendant animals
  • the initial transgenic animal is known as a "founder" animal
  • the founder animal may have the foreign DNA or transgene incorporated in all of its cells or a sufficient proportion such that its progeny stably inhe ⁇ t the transgene Where the transgene is only present in a proportion of cells of the animal, the animal is referred to as a chimera
  • the present invention also extends to animals which incorporate the transgene stably or directly into their chromosomes and which express the transgene m their somatic cells without passing the gene onto their offsp ⁇ ng
  • transgenic in relation to animals, should not be taken to be limited to referring to animals containing in their germ line one or more genes from another species, although many transgenic animals will contain
  • the term refers more broadly to any animal whose germ line has been the subject of the introduction of a warmer-like element (MLE). So, for example, an animal in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a transgenic animal for the purposes of this invention as much as an animal to whose germ line an exogenous DNA sequence has been added.
  • MLE warmer-like element
  • the invention is applicable to all animals, including birds, such as domestic fowl, amphibian species and fish species. In practice, however, it will be to non-human animals (warm-blooded vertebrates), especially (non-human) mammals, particularly placental mammals, and birds, particularly poultry, that the greatest commercial useful applicability is presently envisaged. It is with ungulates, particularly economically important ungulates such as cattle, sheep, goats, water buffalo, camels and pigs that the invention is likely to be most useful. Of the avian species, the invention has particular application to poultry, including domestic fowl Gallus domesticus, turkeys and guinea fowl. It should also be noted that the invention is also likely to be applicable to other economically important animal species such as, for example, horses, llamas or rodents, e.g. rats, mice or rabbits.
  • the method of the present invention is directed towards the introduction of foreign DNA or a transgene into an animal embryo cell.
  • the embryo cell may be at the single cell stage immediately following fertilisation which is the zygote stage.
  • the introduction may be into an embryo cell from a later stage of embryonic development, e.g. from a 2-cell, 4-cell, 8-cell, 16-cell, 32-cell, or 64-cell stage embryo, or from an even later stage.
  • a founder transgenic animal produced from such a later stage embryo may therefore be a chimera but its offspring can be selected for the presence of the transgene in all cells.
  • the manner-hke element may be the transposable element mariner from Drosophila mauritiana or a closely related element from another vertebrate or invertebrate species (Robertson, H M , Nature 362 241 (1993))
  • the manner-hke element may conveniently be de ⁇ ved from the cells of the animal whose chromosomes are to altered
  • a nucleotide sequence which is a mariner-like element can be defined by its ability to act as a transposable element when introduced into a cell
  • Manner-hke transposable elements are about l,300bp long with terminal inverted repeats of about 30bp
  • Each manner-hke element encodes a polypeptide that is a putative transposase and that has, on average, 34% ammo acid sequence identity with the polypeptides encoded by other manner-hke elements
  • the am o acid sequences of the putative transposases of all manner-like elements include a characte ⁇ stic motif known as D, D34D, where "D' represents an aspartate residue The third aspartate of this motif is followed immediately by a tyrosme residue (Robertson, H M , J Insect Physiol 41 99-105 (1995))
  • the transgene can be contained in the manner-hke element at any point withm the mariner sequence Without being bound by theory, it is believed that approximately the final 100 bases of each end of the marine/ -like element may be important for function of the MLE and its incorporation into the chromosomes of a cell Thus the transgene may be positioned anywhere withm in the MLE except less than approximately 100 bases from each end The transgene may also replace the central sequences of the MLE, with only the ends of the element being retained
  • the transgene sequence contained m the manner -like element may be any desired foreign gene sequence
  • Particularly preferred gene sequences include, but are not limited to. those gene sequences coding for proteins which are therapeutically useful, such as enzymes, hormones or other functionally active proteins, e g immunoglobulins, haemoglobin, myoglobm, cytochromes, etc
  • Other gene sequences may encode proteins whose genes are absent or mutated such that the corresponding protein is not produced or is not produced in active form, 1 e. genes responsible for disease conditions such as cystic fibrosis or muscular dystrophy
  • the transgene sequences will also contain promoter sequences to direct expression of the transgene in a selected tissue, e g the mammary gland for secretion in the animals milk, in the yolk or albumen of an egg, or in the blood Further applications, include the expression of regulatory proteins that control immune rejection such that the organs of the transgenic host ammal may be used in Xenotransplantion into a recipient which is allogemc for the immune proteins being expressed m the cells Similarly, allotransplantation is also included
  • transgenes introduced into the animals may include, but are not limited to, disease resistance genes, growth enhancing genes or genes which provide for improved charac ten sitess in a particular trait or introduction of a novel trait
  • MLE manner-hke element
  • the MLE may be introduced m the form of a construct comp ⁇ sing the DNA sequence o ⁇ & manner-hke element and the desired transgene or simply the nucleotide sequence of a manner-hke element and the desired transgene itself may be introduced
  • the construct may be a plasmid, a cosmid or an artificial chromosome such as a Yeast Artificial Chromosome (YAC) or a Bacte ⁇ al Artificial Chromosome (BAC)
  • YAC Yeast Artificial Chromosome
  • BAC Bacte ⁇ al Artificial Chromosome
  • the constructs may also contain additional regulatory sequences, if required, such as promoters or enhancers, depending of the foreign DNA being mtroduced
  • a further aspect of the present invention is therefore a construct or a vector comp ⁇ sing a manner-hke element containing a transgene as desc ⁇ bed above
  • the MLE will be cloned in a plasmid vector for ease of manipulation
  • the method of the present mvention may contain an additional optional step of introducing exogenous transposase protein, or a DNA or RNA sequence encoding a transposase
  • the manner-hke element and the transposase introduced into the cell may be de ⁇ ved from the same ammal species or different species
  • a method for the preparation of a transgenic animal embryo comp ⁇ sing the step of introducing a mannei -like element (MLE) contaimng a transgene into an animal adult cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encoding a transposase
  • MLE mannei -like element
  • a method for the preparation of a transgenic animal embryo comp ⁇ sing the step of introducing a manner-hke element (MLE) contaimng a transgene into an animal foetal cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encodmg a transposase
  • MLE manner-hke element
  • the resulting animal embryo may be prepared by (l) removing the nucleus of the cell following introduction of the manner- ke element containing the transgene and its insertion mto the chromosomes, and (n) subsequently introducing the nucleus mto an enucleated oocyte which is allowed to develop mto an animal embryo.
  • nuclear transfer techniques include, but are not limited to those described in WO-A-9607732, WO-A-9707669 and WO-A- 9707668.
  • a nucleus is transferred from a donor cell to a recipient cell.
  • the use of this method is not restricted to a particular donor cell type.
  • the donor cell may be as described in Wilmut et al Nature 385 810 (1997); Campbell et al Nature 380 64-66 (1996); or Cibelli et al Science 280 1256-1258 (1998). All cells of normal karyotype, including embryonic, foetal and adult somatic cells which can be used successfully in nuclear transfer may in principle be employed in a method according to the present invention.
  • Foetal fibroblasts are a particularly useful class of donor cells.
  • Donor cells may be, but do not have to be, in culture and may be quiescent.
  • Nuclear donor cells which are quiescent are cells which can be induced to enter quiescence or exist in a quiescent state in vivo.
  • Cultured bovine primary fibroblasts, an embryo-derived ovine cell line (TNT4), an ovine mammary epithelial cell derived cell line (OME) from a 6 year old adult sheep, a fibroblast cell line derived from foetal ovine tissue (BLWFl) and an epithelial-like cell line derived from a 9-day old sheep embryo (SECl) are described in WO-A-9707669 and WO-A-9707668.
  • a class of embryo-derived cell lines useful in the invention which includes the TNT4 cell line described in WO 96/07732.
  • Cultured inner cell mass (CICM) cells are described in WO-A9737009 and WO-A- 9827214 and embryonic or stem-like cell lines are described in WO-A-9807841.
  • Transgenic bovine fibroblasts for use as nuclear donors are described in Zawada et al (Nature Medicine 4 (5) 569-574 (1998) and in Cibelli et al (Science 280 1256-1258 (1998))
  • the donor cells are desc ⁇ bed as being quiescent, such cells may not be actively proliferating by means of the mitotic cell cycle
  • the use of a quiescent donor cell is desc ⁇ bed m WO-A-9707669
  • the mitotic cell cycle has four distinct phases, Gl, S.
  • the beginning event in the cell cycle takes place m the Gl phase and has a unique function
  • the decision or commitment to undergo another cell cycle is made at start
  • a cell has passed through start, it passes through the remainder of the Gl phase, which is the pre-DNA synthesis phase
  • the second stage, the S phase is when DNA synthesis takes place
  • the G2 phase which is the pe ⁇ od between DNA synthesis and mitosis Mitosis itself occurs at the M phase
  • Quiescent cells (which include cells in which quiescence has been induced as well as those cells which are naturally quiescent, such as certain fully differentiated cells) are generally regarded as not being m any of these four phases of the cycle, they are usually desc ⁇ bed as bemg in a GO state, so as to indicate that they would not normally progress through the cycle
  • the nuclei of quiescent GO cells have a diploid DNA content
  • Cultured cells can be induced to enter the quiescent state by various methods including chemical treatments, nut ⁇ ent depnvation, growth inhibition or manipulation of gene expression
  • the cells exit the growth cycle dunng the Gl phase and arrest, as explained above, m the so-called GO stage
  • Such cells can remain m this state for several days (possibly longer depending upon the cell) until re- stimulated when they re-enter the growth cycle
  • Quiescent cells arrested in the GO state are diploid
  • the GO state is the pomt m the cell cycle from which cells are able to differentiate
  • On quiescence a number of metabolic changes have been reported and these include monophosphorylated histones, ciliated cent ⁇ oles, reduction or complete cessation m all protein synthesis, increased proteolysis, decrease m transc ⁇ ption and increased turnover of RNA resulting m a reduction m total cell RNA, disaggregati
  • the recipient cell to which the nucleus from the donor cell is transferred may be an oocyte or another suitable cell.
  • a preferred class of recipient oocyte is described in WO-A-9707668.
  • Recipient cells at a variety of different stages of development may be used, from oocytes at metaphase I through metaphase II, to zygotes and two-cell embryos. Each has its advantages and disadvantages.
  • the use of fertilized eggs ensures efficient activation whereas parthenogenetic activation is required with oocytes (see below).
  • Another mechanism that may favour the use of cleavage-stage embryos in some species is the extent to which reprogramming of gene expression is required. Transcription is initiated during the second cell cycle in the mouse and no major changes in the nature of the proteins being synthesised are revealed by two- dimensional electrophoresis until the blastocyst stage (Howlett & Bolton J Embryol. Exp. Morphol.
  • the recipient cells will be oocytes. It is preferred that the recipient be enucleate While it has been generally assumed that enucleation of recipient oocytes in nuclear transfer procedures is essential, there is no published expe ⁇ mental confirmation of this judgement
  • the o ⁇ gmal procedure desc ⁇ bed for ungulates involved splitting the cell mto two halves, one of which was likely to be enucleated (Willadsen Nature 320 (6) 63-65 (1986)) This procedure has the disadvantage that the other unknown half will still have the metaphase apparatus and that the reduction in volume of the cytoplasm is behe ⁇ ed to accelerate the pattern of differentiation of the new embryo (Eviskov et al Development 109 322-328 (1990))
  • a fourth aspect of the present invention there is provided a method for the preparation of an ammal, the method comp ⁇ sing the steps of (a) prepanng an embryo according to any of the preceding aspects of the present invention,
  • ammal embryo prepared m accordance with this aspect of the present invention may be further manipulated p ⁇ or to full development of the embryo This may include the introduction of additional genetic matenal or to assay the embryo for particular genetic characte ⁇ stics or the presence or absence of a gene It is also possible that more than one ammal can be denved from the embryo where the cells of the embryo are used to prepare more than one embryo allowed to develop to term
  • the present invention therefore also extends to an ammal prepared by a method according to the fourth aspect of the invention
  • a manner-like element m the therapy of a disease condition caused by the absence of a gene or the mutation of a gene
  • This aspect of the invention also extends to the use of a manner-like element m the preparation of an agent for the prophylaxis or treatment of a disease caused by the absence of a gene or the mutation of a gene
  • Such methods of treatment may compnse the introduction of a manner-hke element contaimng a transgene mto an ammal cell Where transposase protein, or a DNA or RNA sequence encoding transposase is also to be introduced, this step may be simultaneous, sequential or separate to the introduction of the MLE
  • the present invention therefore also extends to the preparation of an embryo according to any one of the preceding aspects of the invention m which the cells of the embryo are used in the treatment of a disease condition associated with the absence of a gene or the mutation of a gene Such cells may also be used to treat disease conditions in which the patient's cells are no longer active or effective especially neurological or hormonal disorders
  • Preferred features for the second and subsequent features are as for the first aspect mutatis mutandis
  • FIGURE 1 shows PCR analysis of DNA extracted from embryos and chicks that survived for at least 12 days of incubation after injection of the r ⁇ rzMer-containing plasmid pMosl
  • FIGURE 1(a) shows a diagram of pMosl, indicating sequences identified by PCR and unique rest ⁇ ction sites
  • FIGURE 1(b) shows a graphical presentation of the results given m Table 1
  • the results of injecting pMosl with and without the addition of recombinant-de ⁇ ved transposase protein are compared
  • FIGURE 2 shows a Southern blot analysis of genomic DNA isolated from individual Gi transgenic chicks, hybridised with a manner probe
  • FIGURE 2(a) shows BamHI/Hindlll digests of individual chicks which each have a novel pattern of m ⁇ «er-hyb ⁇ d ⁇ s ⁇ ng fragments (lanes 1 to 7), and of the parent Go cockerel (lane 8)
  • a control digest of non-transgemc chick DNA was run in lane 9
  • FIGURE 2(b) shows EcoRI digests of samples from the same birds as in FIGURE 2(a)
  • the Band arrowed in lane 2 is the EcoRI fragment cloned in pZAP13 (see FIGURE 3)
  • FIGURE 3 shows the characte ⁇ sation of a single integrated marinei element
  • FIGURE 3(a) shows the Southern blot of genomic DNA from individual Gi chicks digested with BamHI and Hindlll (from FIGURE 2(a)) was stripped and reprobed with the EcoRI insert from pZAP13. Hybridisation to a range of restriction fragments can be seen in all the samples, including the negative control (lane 9).
  • FIGURE 3(b) shows a comparison of the sequence across the left and right ends of the mariner element in pMosl and pZAP13.
  • FIGURE 4 shows PCR analysis for present of the Tet R gene in DNA from embryos and chicks that survived for at least 12 days of incubation after introduction of pMoslTet.
  • the copy number of the Tet R gene was estimated as described in the "Materials and Methods" and the results of co- injection of transposase protein compared with injection of plasmid alone.
  • Example 1 Preparation of Transposase The mariner transposase used in the following experiments was purified from E. coli strain BL21 DE3 (Studier et al Methods in Enzymology 185 60-89 (1991)) carrying the plasmid pBCPMosl. This was derived from the expression vector pBCP368 (Velterop et al Gene 153 63-65 (1995)). The complete coding sequence of mariner transposase from the element Mosl was inserted at the Ndel site of pBCPMosl.
  • the extract was pipetted up and down a few times until the viscosity decreased and was then left at room temperature for 10 minutes
  • the whole cell extract was then centnfuged at 20,000g for 30 minutes
  • the pellet was washed three times in 0 5% NP40 (v/v), ImM EDTA and followed by one wash in 6M urea before being finally being resuspended m 1ml of 25mM Tns-HCl (pH7 5), 6M guamdme hydrochlo ⁇ de, 5mM DTT
  • the supernatant was diluted one hundred-fold mto 25mM Tns-HCl (pH7 5), 8M urea, 5mM DTT, 10% glycerol buffer and loaded onto a 2ml fast flow CM Sepharose column (Sigma) pre-equihbrated with the same buffer supplemented with 50mM NaCl
  • Plasmid Mosl contaimng the mariner element (Medhora et al Genetics 128 311-318 (1991)) w as injected mto chicken embryos as desc ⁇ bed by Sang & Perry (Mol Reprod Dev 1 98-106 (1989)) at a concentration of 25 ⁇ g/ml together with pu ⁇ fied mariner transposase at a concentration of 0 05-0 005mg/ml m a buffer contaimng lOOmM NaCl, 25mM HEPES pH7 7, 2mM dithiothreitol, 5% (v/v) glycerol, 25 ⁇ g/ml bovine serum albumin, with or without 5mM Manganese acetate
  • the injected embryos were cultured as desc ⁇ bed by Perry (Nature 331 70-72 (1988)) Hatched chicks which had cells contaimng mariner sequences were identified by carrying out polymerase chain reactions (PCR) with primers specific to mariner
  • the plasmid pMoslTet was constructed by insertion of the tetracyclin resistance gene into the unique Sail site present in the open reading frame of mariner in Mosl.
  • the Tet gene was obtained by digestion of pBR322 with Aval and EcoRI, pMosl was linearised with Sail and the two fragments ligated after treatment with Klenow polymerase to fill in the ends.
  • the expression and preparation of recombinant-derived mariner transposase will be described in detail elsewhere (A. Dawson and D. Finnegan, in preparation).
  • the mariner transposase gene from pMosl was inserted into the expression vector pBCP368 (Velterop et al Gene 153 63-65 (1995)) to generate the construct pBCPMosl .
  • This construct was transferred into E. coli strain DH5 ⁇ and the cells harvested after induction of protein expression.
  • the transposase protein was recovered as an insoluble precipitate, solubilised and bound to fast flow CM sepharose column (Sigma). The protein was renatured in 8M urea and the activity measured in an in vitro transposition assay.
  • Chick embryo culture was essentially as described (Perry Nature 331 70-72 (1988)) with modifications noted in (Love et al Bio/Technology 12 60-63 (1994)). Between 1 and 2nL of uncut plasmid, at a concentration of 25 ⁇ g/ml, was injected into the germinal disc of zygotes following established procedures (Love et al Bio/Technology 12 60-63 (1994)).
  • transposition buffer (lOOmM NaCl, 25mM HEPES pH7.9, 2mM dithiothreitol, 50mM manganese acetate, 25 ⁇ g/ml BSA, 5% glycerol) and transposase protein added when required to a concentration of 15ng/ml.
  • Tissue samples (chorioallantoic membrane, liver and gonads) were dissected from embryos which died in culture after more than 12 days of incubation and DNA extracted using Puregene (Flowgen) genomic DNA purification kit. Genomic DNA samples were obtained from chorioallantoic membrane at hatch of surviving chicks, blood samples from older birds and semen from the mature cockerel. PCR analysis was carried out on 0.5-l ⁇ g DNA samples for the presence of the mariner element and pBluescript (pMosl experiments) or for Tet R gene and the vector chloramphenicol (CAT) resistance gene (pMoslTet experiments).
  • CAT vector chloramphenicol
  • Control PCR reactions were carried out in parallel on l ⁇ g aliquots of chicken genomic DNA with pMosl or pMoslTet DNA added in quantities equivalent to that of a single copy gene (IX) a 10-fold dilution (0.1X) and a 100- fold dilution (0.01X) as described previously (Love et al Bio/Technology 12 60-63 (1994)).
  • the primers used were:
  • ⁇ rz ..er-hybndismg fragments present in chick 13 genomic DNA and found to co- migrate with the approximately 8kb fragment
  • the pZapl3 clone was sequenced using pnmers near the 5' and 3' end of mariner, designed to sequence across the ends of the element mto the flanking genomic DNA
  • Example 5 Transposition of mariner into the chicken genome
  • the probe identified a series of EcoRI restriction fragments in all the chicken genomic DNA samples, including DNA from a wild-type chick ( Figure 3(a), lane 9).
  • the mariner hybridising fragments are also faintly detectable.
  • the clone was also analysed by DNA sequencing, using pnmers internal to the ends of mariner, designed to prime sequence over the ends of the inserted mariner element, if complete.
  • the sequence generated ( Figure 3(b)) corresponds exactly to the sequence of the ends of the mariner element but is flanked by sequences that differ from the Drosophila genomic DNA adjacent to the element in pMosl.
  • the element present in the chicken DNA is flanked by TA dinucleotide repeats, the sequence ' characteristically generated by mariner transposase-mediated transposition.
  • the transposase activity could have been due to expression of the Mosl transposase gene or an endogenous activity present in the chick zygote.
  • a series of zygote injection experiments was carried out using a construct in which the mariner transposase gene was inactivated by insertion of the tetracyclin resistance gene (Tet R ) within the transposase coding region (pMoslTet).
  • Tet R tetracyclin resistance gene
  • pMoslTet transposase coding region
  • Example 7 Germline Stability of Integrated Mariner Elements Two Gi birds, cockerels 3 and 7, that each had a single copy of mariner integrated at different chromosomal sites ( Figures 2(a), lanes 4 and 5) were selected to analyse stability of the elements after germline transmission to the G 2 generation. They were each crossed with stock hens, DNA extracted from resulting embryos and screened by PCR to identify transgenic embryos.
  • the ratios of transgenic to non-transgemc offspnng from cockerel 3 (65 59) and cockerel 7 (64 57) did not differ significantly from the expected 1 1 Mendehan ratio
  • the genomic DNA from transgenic embryos was digested with BamHI and Hmdlll and the pattern of mariner-hybndismg fragments compared to the single band present in the transgenic parent
  • All of the transgenic offspnng from both cockerels had a single mariner band that co-migrated with the restnction fragment present in the parent cockerel (data not shown) There is no evidence of instability of mariner after transposition, although a low level of instability would not have been detected
  • pMosl An intact plasmid construct (pMosl), will be injected mto the pronucleus or the cytoplasm of mouse fertilised eggs at a concentration of approximately 1 5ng/ ⁇ l
  • the method used is as descnbed by Whitelaw et al (Biochemical J 286 31-39 (1992)) and is based on the work of Bnnster et al (P oc Nat'l Acad Sci USA 82 4438-4442 (1985)) In some expenments recombmant-denved, pu ⁇ fied marine! transposase protein or mRNA.
  • mice will be included The mouse embryos will be transferred to surrogate mothers and new-born mice will be screened to identify any transgenic for mariner All transgenic mice will be analysed further to determine if the manner element is present in the mice as a result of transposase- catalysed transposition or random integration of the whole plasmid construct
  • the frequency of mariner transposition mto the chicken genome indicated by this analysis is high (over 20%), although this has to be confirmed by the generation of additional transgenic birds
  • the proportion of Gi birds that inhented manner from the Go cockerel was approximately 30%, a 10-fold higher transmission frequency than obtained after introduction of linear gene constructs (Love et al Bio/Technology 12 60-63 (1994))
  • the analysis of Gi birds indicated that there had been multiple insertions of mariner Two possible explanations cannot yet be distinguished between either that several independent transposition events took place from the introduced plasmid or that a copy of mariner transposed into the chicken genome and that this was followed by secondary transposition events
  • the fact that two copies of mariner w ere stably transmitted to the G 2 generation suggests that, once integrated mto

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Abstract

L'invention concerne un procédé pour créer un embryon d'animal transgénique. Ce procédé consiste à introduire, dans une cellule embryonnaire de l'animal, un élément semblable au Mariner (MLA) qui comprend un transgène, et éventuellement à introduire une protéine transposase exogène ou une séquence d'ADN ou d'ARN codant pour une transposase. On peut utiliser l'embryon qui résulte de cette opération pour produire de nouveaux embryons ou pour lui permettre de se développer et donner un animal. L'invention peut servir à introduire des ADN étrangers dans des animaux sélectionnés.
EP98939777A 1997-08-22 1998-08-21 UTILISATION DE L'ELEMENT $i(MARINER) POUR CREER DES ANIMAUX TRANSGENIQUES Withdrawn EP1006790A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GBGB9717913.9A GB9717913D0 (en) 1997-08-22 1997-08-22 Dna
GB9717913 1997-08-22
GBGB9812822.6A GB9812822D0 (en) 1998-06-12 1998-06-12 Dna
GB9812822 1998-06-12
PCT/GB1998/002517 WO1999009817A1 (fr) 1997-08-22 1998-08-21 Utilisation de l'element mariner pour creer des animaux transgeniques

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EP1006790A1 true EP1006790A1 (fr) 2000-06-14

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US6299850B1 (en) 1999-03-16 2001-10-09 The United States Of America As Represented By The Department Of Energy Carbon activation process for increased surface accessibility in electrochemical capacitors
JP2003502015A (ja) * 1999-03-17 2003-01-21 パラダイム ジェネティックス、 インコーポレイテッド 生物中における遺伝子ノックアウトライブラリの迅速かつ大量産生のための方法類および素材類
WO2001029205A2 (fr) * 1999-10-19 2001-04-26 Minos Biosystems Limited Procede de manipulation genetique
CA2402924A1 (fr) * 2000-03-21 2001-09-27 Charalambos Savakis Procede de production d'organismes transgeniques a l'aide de transposons
JP2004267001A (ja) * 2000-08-16 2004-09-30 Kansai Tlo Kk トランスジェニック哺乳動物及びその作製方法並びにこれを用いた遺伝子機能の解明方法
US20030092179A1 (en) * 2001-09-24 2003-05-15 Patrick Fogarty Animal integration vector and methods for its use
DE60329366D1 (de) 2002-01-09 2009-11-05 Univ Erasmus Medical Ct Induktierbare transposition beim transgenen organismus mit transposon-vektor
US20040172667A1 (en) 2002-06-26 2004-09-02 Cooper Richard K. Administration of transposon-based vectors to reproductive organs
BR0305217A (pt) * 2002-06-26 2005-06-28 Transgenrx Inc Regulação gênica em animais transgênicos usando um vetor baseado em transposon
FR2850668B1 (fr) * 2003-01-31 2005-04-08 Centre Nat Rech Scient Elements genetiques mobiles appartenant a la famille mariner chez les eucaryotes hydrothermaux
WO2005003359A1 (fr) * 2003-07-08 2005-01-13 Japan Science And Technology Corporation Procede et systeme permettant de mettre au point un organisme transgenique
WO2005062881A2 (fr) 2003-12-24 2005-07-14 Transgenrx, Inc. Therapie genique faisant intervenir des vecteurs de transposon
WO2010036978A2 (fr) 2008-09-25 2010-04-01 Transgenrx, Inc. Nouveaux vecteurs pour la production d'hormone de croissance
WO2010036976A2 (fr) 2008-09-25 2010-04-01 Transgenrx, Inc. Nouveaux vecteurs pour la production d'anticorps
EP2417263B1 (fr) 2009-04-09 2015-09-23 ProteoVec Holding L.L.C. Production de protéines au moyen de vecteurs à base de transposon
BR112022018442A2 (pt) 2020-03-15 2022-11-22 Proteinea Inc Produção de proteína recombinante em insetos

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GB8906214D0 (en) * 1989-03-17 1989-05-04 Nat Res Dev Introducing an exogenous gene into birds
GB9517780D0 (en) * 1995-08-31 1995-11-01 Roslin Inst Edinburgh Biological manipulation
AU731615B2 (en) * 1996-02-09 2001-04-05 Het Nederlands Kanker Instituut Vectors and methods for providing cells with additional nucleic acid material integrated in the genome of said cells

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