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EP0258427A1 - Procede pour transferer des substances organiques a des cellules-oeufs d'animaux, et compositions utilisees a cet effet - Google Patents

Procede pour transferer des substances organiques a des cellules-oeufs d'animaux, et compositions utilisees a cet effet

Info

Publication number
EP0258427A1
EP0258427A1 EP87902077A EP87902077A EP0258427A1 EP 0258427 A1 EP0258427 A1 EP 0258427A1 EP 87902077 A EP87902077 A EP 87902077A EP 87902077 A EP87902077 A EP 87902077A EP 0258427 A1 EP0258427 A1 EP 0258427A1
Authority
EP
European Patent Office
Prior art keywords
sperms
substances
vesicles
granulae
binding
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.)
Withdrawn
Application number
EP87902077A
Other languages
German (de)
English (en)
Inventor
Oswald Rottmann
Paul HÖFER
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.)
TRANSGENE GmbH
Original Assignee
TRANSGENE GmbH
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 TRANSGENE GmbH filed Critical TRANSGENE GmbH
Publication of EP0258427A1 publication Critical patent/EP0258427A1/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Withdrawn legal-status Critical Current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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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)

Definitions

  • the invention relates to a method of transferring organic and/or inorganic substances to egg cells and/or somatic cells of animals, compositions suited for use in the method, precursors of such compositions, and transgenic animals obtained by the method.
  • Disclosed herein is a method of transferring organic and/or inorganic substances by means of sperms to the respective target cells.
  • the substances to be transferred are protected from dilution, enzymatic or other modification by being entrapped in vesicles or granulae.
  • the envelopes are then bound to sperms.
  • the sperms carry the vesicles or granulae to the target cell, preferably an egg cell, penetrating the outer membranes thereof. Vesicle contents or granula components are released within the cell where they exert their specific effects.
  • the method is particularly suited for transferring nucleic acids into egg cells so as to produce transgenic organisms as well as for studies of teratology and gene regulation in the early stages of embryogenesis.
  • the injection procedure does however require highly sophisticated instruments and extreme skill. Factors influencing integration of the injected gene are: DNA concentration, type of buffer solution, injected cell, form of plasmid , and origin of the embryo, (strain of mice). Length of the DNA molecule (0.5 to 10.0 kb ) , on the other hand, is without influence on integration frequency.
  • DNA of the desired type was injected into the cytoplasm of the zygote, the blastomeres of the bicellular organism, or the blastocoele of the blastocyst. This method has less chance of success, and formation of mosaics is very likely.
  • nucleus replacement A very inefficient method of producing transgenic animals is nucleus replacement.
  • the nuclei of totipotent cells transfected in vitro must be isolated by micromanipulation, the plasma membrane being removed together with the major portion of the cytoplasm.
  • the isolated nucleus is then transferred with a micropipette to a freshly fertilized egg.
  • the nucleus is deposited, and female as well as male pronucleus are removed by suction when the pipette is withdrawn from the egg.
  • the egg cell now has a diploid nucleus containing a (previously in vitro transfected) gene. Extreme skill in the techniques of micromanipulation is required for this method, too, which, though being costly, provides little chances of success.
  • teratocarcinoma cells are transfected and subsequently transferred as gene vehicles into embryos (blastocysts). Chimeras with one transgenic cell line are obtained in this manner.
  • the problems regarding selectivity of expression and diploidy of the desired cells are, however, substantial, and transmission into the germline is a mere coincidence.
  • the genetically manipulated retrovirus used in a further transfer method is capable of infecting embryos. As potential expression of retroviral native genes cannot yet be eliminated reliably, the risk involved in manipulating viruses precludes their use in practice.
  • the artificially inseminated animals must undergo surgery that the embryos may be recovered by flushing. Cows are the only animals where this may be done without an operation but the person conducting flushing needs special training.
  • a catheter is placed near the end of the uterus and sealed against the cervix with a rubber bulb. Buffer solution is then pumped into the uterus, and the embryos will be washed cut when this solution is removed again by suction; the embryos are so tiny that they can only be detected with a microscope.
  • the remaining uninjured embryos may now be retransferred into a foster mother. With cows, this may again be accomplished by non-surgical means but the receiver animal must be hormonally synchronized by elaborate preparatory treatment to adjust the physiological conditions in its uterus to those of a pregnant animal. For pigs and sheep, an operation is again required (cost and time-consuming) for transferring the embryos to the animal that is to carry them to the full term. The young finally born are but a small percentage of the originally available embryos. Of these young ones, a maximum of 25 % (mice) and less than 0.1 to 1 % of sheep, pigs and rabbits are actually transgenic.
  • the method of the invention now serves for incorporating specific substances into animal cells, the object being a modification of the respective cells or investigation of the effects exerted within the cells by certain substances.
  • the method of the invention is based on the long-approved biological principle of DNA transfer, namely use of sperms as transfer vehicles. It also uses the firmly established routine of artificial insemination (particularly of domestic animals), a detailed discussion of which is not necessary here.
  • sperms are advantageously incubated for a short period of time together with synthetically prepared vesicles or granulae which are adsorbed on the sperms under the chosen conditions.
  • the vesicle-loaded sperms are subsequently deposited at the fertilization site where they may enter into the egg cells.
  • the vesicles are prepared in the presence of nucleic acids coding for one or more genes to be transferred, or in the presence of other substances that are to be transferred into egg cells or somatic cells.
  • the substances to be transferred become .entrapped therein, thus being protected during transport to the egg cells .
  • Sperms then take the vesicles into the cytoplasm of the target cells.
  • the granulae dissolve, or the vesicles undergo lysis with cell-inherent enzymes, releasing the entrapped substances.
  • the new gene ( s ) may be present in all cells of the developing organism. Once established in the germinal cell, the genes thus transferred will be transmitted to all descendants of the respective organism.
  • a central aspect of the invention is the following: Sperms transfer substances bound to their surface, e.g. into egg cells. To make transportation by sperms possible, the respective substance must be attached to the sperm surface. It must also be entranced so as to prevent dilution or modification through environmental factors during transport.
  • the active substances are therefore provided with a suitable envelope, the envelope being then attached to the sperm surface. Materials used for this purpose must be capable of firmly connecting sperm surface and envelope. On contact with a target cell, the sperm enters the cell, releasing the transferred substances within the cell interior.
  • the envelopes for the substances to be transferred may always be prepared in accordance with analogous techniques.
  • glycoproteins on the surface of epididymal sperms of rats have shown that a certain type of glycoprotein is present only in the plasma membrane of the tail portion.
  • This and several other types of sperm-covering glycoproteins secreted in the male genital tract are similar to fibronectins, a large group of adhesive glycoproteins found on cell surfaces, in connective tissue and some body fluids. All of these glycoproteins are associated with membranes and play a part in cellular adhesion.
  • spermal membranes are also capable of binding a number of steroid hormones and so-called "second messengers" such as cyclic AMP.
  • Bovine sperms absorb a variety of steroids, of which progesterone forms the strongest bond, followed by oestradiol, dihydrotestosterone, androstanediol and eticholanone.
  • a further component of the outer plasma membrane of sperms is the so-called membrane-bound, substrate-binding transport protein (carboglutelin). This protein binds and absorbs a variety of sugars from the liquid surrounding the sperms, thus supplying the sperms with the required energy.
  • sperms In some cases (gene substitution, somatic gene therapy) where substances are to be transferred by sperms to somatic cells, it might be advisable to first eliminate the haploid genome of the sperms by ionizing radiation so as not to introduce additional genetic material into the diploid target cell.
  • Use of sperms as "genome-free" transfer vehicles is possible, especially if the sperms have first been subjected to high-intensity radiation of short wavelength.
  • the surfaces of biological structures such as the outer plasma membranes of sperms, show external three-dimensional structural elements (groups of molecules) that protrude from the surface (membrane matrix).
  • the membrane as such (basic structure) is composed of a lipid double layer, both surfaces of that layer being hydrophilic.
  • Anchored to this lipid double layer are, among other components, various types of proteins through their lipophilic amino acid sequences.
  • Hydrophilic side chains of proteins and lipids protrude from the surface; they usually are oligo- or polysaccarides or hydrophilic amino acid sections of proteins.
  • These elements are of a special three-dimensional structure and further characterized by electric charges, hydrophilic or hydrophobic interaction, van der Waals' forces, and chemico-physical parameters of the environment. Many of these "protruding" elements are suited as binding points for substances approaching the plasma membrane from the outside. Similar spatial structural elements are found on the surface of the casings used in the method of the present invention.
  • the method of the invention is based on the concept that certain protruding three-dimensional structural elements on the surfaces of sperms might be bound to structural elements on the surface of the envelopes.
  • connection with the plasma membrane of the sperms may result in addition (adsorption) or, when the two units merge, in fusion and resulting endocytosis of the envelope contents.
  • Sperms constitute the substantially "invariable" element in the method of the invention as they vary only in accordance with their state of maturation and the species from which they were obtained; in other words: depending on the species, sperms show a more or less differently composed surface structure.
  • binding suited for the method of the invention may be divided in two groups:
  • a structural element on the surface of an envelope corresponds sterically (as a structure in space) as well as physicochemically (e.g. opposite charge) to an elevated structural element ("key") on the sperm membrane (key-lock principle).
  • key an elevated structural element
  • Phenomena of a purely physical nature such as oppositely charged sperm surfaces and envelopes, also result in direct binding.
  • specific virus receptors may be mentioned that bind to precisely definable structural elements on the sperm surface.
  • Elements of the envelope virus membrane proteins, i.e.
  • virus receptors serve as a lock-type mechanism and permit anchoring of complete viruses (envelope protein with genome) to certain surface characteristics of the sperms (key).
  • spermspecific virus receptors may be incorporated in the envelopes (particu arly membrane-type envelopes) to serve as binding molecules for the sperms. Similar receptors are encountered in various microorganisms such as certain mycoplasms and bacteria. Covalent bonding of sperm-specific molecules such as antibodies and lectins, to components of the envelopes is possible , too. These molecules serve the same functions in binding as the above mentioned virus receptors. Blends of different binding molecules may also be used.
  • the adhesive as such may comprise either two or more "locks", or a "key” plus one or more "locks”.
  • a simple form of binding may be obtained with positively charged ions (Fe ++ , Fe +++ , Zn ++ , and many others) located between negatively charged surfaces of sperms and envelopes.
  • positively charged ions Fe ++ , Fe +++ , Zn ++ , and many others
  • disadvantage may be certain instabilities due to the unspecific nature of binding by charge and probably due to chelate complex-forming substances that frequently neutralize the charged ions.
  • lectins are characterized by highly specific binding capacity and sometimes extremely high binding affinity for certain sugar molecules. Hardly ever is there recognized a group other than the terminal sugar group of an oligo- or polysaccharide chain which, in turn, is covalentiy bonded to a lipid molecule (glycolipid) or a protein molecule (glycoprotein). Some lectins always bind to a certain type of sugar molecule under the chosen conditions while others are capable of binding different types of sugars. As the lectin molecule has two or more binding sites (bivalent or polyvalent), lectins are capable of adhesively connecting surfaces showing identical terminal sugar groups.
  • binding capacity may also be applied to sperms.
  • Incubation of sperms (in diluted suspension) with an excess of Con A results in saturation of all Con A-binding sites on the sperm membrane.
  • the surface of the sperms is subsequently covered by a large number of protruding free sugar binding sites through which any envelope showing the corresponding sugar groups (alpha-mannose and glucose, respectively) on its surface may be bound to the sperms.
  • binding molecules are, due to their bivalent or polyvalent properties, equally suited for the above technique of oversaturating the envelopes and subsequently binding them to sperms.
  • Use of spermal antigen-specific antibodies requires the presence of spermal antigen on the envelope surfaces. This may most easily be accomplished by isolating the plasma membranes of sperms and using them as envelopes.
  • Spermal antigenspecific antibodies and antisera respectively, may be recovered from the serum of female animals after repeated immunization with washed sperms.
  • Optimum binding capacity must be attributed to spermal antigen-specific, high affinity monoclonal IgM antibodies as they have ten binding sites ( decavalent). Binding strength may approach that of covalent bending.
  • hybrid anti-bodies In contrast to naturally occuring antibodies, they may have two or more binding sites for recognizing and binding different structural elements. Thus, envelopes maybe bound to sperms even if the structural surface elements of envelopes and sperms are not identical. Similar "unequal" binding is possible with a protein (protein A) found in the cell walls of Staphylococcus aureus and normal spermal antigen-specific antibodies. Protein A is capable of specifically binding with high affinity to that part of an antibody that does not bind antigen (F part).
  • protein A protein found in the cell walls of Staphylococcus aureus and normal spermal antigen-specific antibodies. Protein A is capable of specifically binding with high affinity to that part of an antibody that does not bind antigen (F part).
  • envelopes thus coated with protein A and antibodies are capable of binding very specifically and firmly to sperms.
  • Another phenomenon based on non-specific adsorption of proteins on surfaces may be utilized for forming very strong specific bonds between envelopes and sperm surfaces. This so-called “coating” (covering) of surfaces is made possible by the high non-specific bind ng capacity of proteins for certain surfaces such as cell memb anes, synthetics or glass.
  • sperm-specific antibodies are used for covering the envelopes (vesicles, especially liposomes).
  • "Coating” may be effected with spermal antigen-specific monoclonal antibodies, purified polyclonal antibodies or with an easily prepared high-titer anti- serum. Envelopes covered with such specific binding molecules show the following advantages:
  • the specific binding structures may be stabilized by adding bond- promoting molecules (e.g. albumin, transferrin).
  • bond- promoting molecules e.g. albumin, transferrin
  • envelopes of the substances to be transferred by sperms prote their contents from enzymatic or other modification and bind the to the sperms.
  • envelope refers to any system which eithe entraps the substances to be transferred or is capable of formin aggregates therewith.
  • envelopes Preferably used as envelopes are vesicular structures, the substances to be transferred being either contained in the lumen thereof as an aqueous suspension or, if they are lipophilic, integrated in the walls of these vesicles.
  • the walls of such vesicles may consist of lipids or lipid-type materials that show high stability in aqueous media. Suited for aggregation are materials permitting direct addition (without lumen) to the substances to be transferred without denaturing these substances. With the aid of calcium phosphate or histone protein, granulae may thus be prepared from e.g. nucleic acids.
  • the binding form must not negatively affect the ability of the sperms (or parts thereof) to fuse with the target cells.
  • the surfaces of some microorganisms and viruses are provided with receptors permitting specific binding to sperms.
  • a virus specialized on sperms of fish infects the egg cells by becoming attached to the sperm surface, thus finding its obligatory host.
  • virus membranes or cell walls of microorganisms are in themselves a sort of envelope, they seem perfectly suited for any transfer method involving sperms. But they can take up only a limited number of substances and are technically more difficult to handle (isolation, reaggregation of components) than the natural or artificial lipid membranes described hereinafter.
  • Certain receptors from the membranes of viruses or microorganisms may be integrated into lipid membranes, serving therein as structural elements of specific binding affinity for sperms (virus receptors, protein A and the like). Lipid membranes may be recovered without problems from substantially any animal cell.
  • the only important aspect is that the selected cells or plasma membranes show surface characteristics that permit direct or indirect binding to the surface of sperms. This depends primarily on the desired form of binding.
  • Sugar-binding lectins for instance, may serve as mediator molecules between sperms and lipid membrane, and erythrocyte membranes (so-called "ghosts”) may be used in this case as an easily obtainable starting substance as they exhibit a plurality of protruding sugar groups.
  • composition of the outwards extending structural elements of the lipid membrane may be modified so as to create new or better conditions for the desired form of binding between sperms and envelopes.
  • Terminal sialinic acid molecules may be removed by cleavage, the lipid vesicles being incubated (before or after the substances to be transferred have been entrapped therein) with neuraminidase to effect cleavage.
  • the resulting reduction in negative surface charge as well as the many previously hidden sugar molecules thus exposed offer new ways of binding the vesicles to sperms.
  • Another possibility of optimizing special binding forms is use of enzymes capable of cleaving sugars as well as proteins. Even "recoating" of the lipid membrane surface may be accomplished by enzymatic treatment methods. Of particular interest in this respect are glycosyltransferases which covalently bond certain sugar molecules to the sugar backbone of the lipid membrane, thus making other forms of binding possible.
  • sperm-specific antibodies are used as binding mediators; best suited for this purpose are envelopes prepared from plasma membranes of sperms. Due to identical antigenic structural elements on sperms and envelopes, the two units can easily be connected to each other through bivalent or polyvalent antibodies.
  • the biochemical preparation method of such biological membranes prevents transfer of undesirable substances such as common viruses.
  • Lipid membranes of the above type may also be set up artificially from the respective lipids. Molecules (cerebrosides, glycoproteins and the like) must however be incorporated in the lipid mixture, their structural elements protruding from the surface permitting direct or indirect binding to sperms.
  • organic and/or inorganic substances are also suited for entrapping the substances to be transferred.
  • a form of granular envelope, particularly suited for nucleic acids, is obtained by calcium phosphate precipitation of DNA. What physico-chemical properties actually make binding of calcium phosphate-precipitated DNA to cell membranes possible is not yet fully understood, but the method as such is practicable.
  • a natural type of granular envelope for DNA of higher organisms is obtained by binding DNA to histone proteins.
  • the histone envelope serves a stabilizing function and provides some protection from degradation to the DNA.
  • the histone-associated 5NA is advantageously incorporated in vesicles as they show far better binding characteristics and offer added protection.
  • the method of the invention may serve three objects:
  • a plurality of substances may be transferred according to the method of the invention. Due to the proposed technique of simultaneously preparing envelopes and enclosing therein the substances to be transferred, the most diverse substances may be entrapped in the same manner and without the need for specially developed processing steps. Binding to sperms and thus transfer to the target cells is not dependent either on the type of substance within the envelopes.
  • Transgenic organisms or transgenic cells can be obtained only by transferring genetic material in form of nucleic acids.
  • a plurality of different genes useful for improving production are available at present or will become available in the near future.
  • Of particular interest are: the gene for somatotropin (growth hormone), genes for various milk proteins (caseins), use of anti-sense DNA for controlling specific diseases, and use of genes for producing vaccines in large quantities (genetic farming).
  • the invention makes use of the phenomena of natural fertilization (fusion of sperms and eggs) and of artificial insemination, procedures (manipulative transfer of sperm) to provide a significantly simplified method of transferring genes to organisms wherein conventional techniques such as embryo flushing, micromanipulation, surgery and transfer of embryos are avoided.
  • the procedure by which the desired substances (genes) are prepared is substantially the same as in prior methods of producing transgenic organisms.
  • Recommended is use of special integration and expression enhancers ( metailothioneine promoters, long terminal repeats) to facilitate integration and expression of the genes thus transferred.
  • Nucleic acids (genes) in linear or circular form are either entrapped in artificial vesicles or processed with specific proteins and inorganic salts, respectively, to form granulae.
  • tracers may be added and transferred within the same envelopes.
  • Illustrative of such tracers are small genome units that undergo self-replication in the course of embryonal development (episomal Bovina papilioma virus and many others); presence of such units in embryos is relatively easy to verify by means of DNA hybridization techniques. Testing in the above manner may be conducted not only in vitro but in vivo as well without involving the organism of the foster mother. Enzymatic transformation of many substances by the maternal organism frequently prevents in vivo investigation of embryopathogenesis.
  • sperms may serve as target cells for the substance-loaded sperms: the natural target cells of sperms, i.e. egg cells, and all other cell types of multicelluiar organisms, including transformed ceils such as teratocarcinoma cells.
  • Egg cells of many invertebrates, fish and amphibia are extracorporally accessible (spawning).
  • the egg cells of most higher vertebrates lie more or less hidden in the primary reproductive organs (sexual organs) of the female. They are accessible only by surgical means.
  • Sperms alone are capable of reaching the egg cells within the sexual organs of the female and transferring (aided by the method of the invention) additional substances to these egg ceils.
  • Bovine sperm covers the long distance between the site of sperm deposition (natural as well as artificial insemination) near the cervix and the ovarial end of the fallopian tube within a few minutes.
  • the slow proper motion of the sperms (0.2 mm/sec.) is strongly enhanced by ciliary motion and the activity of secretory fluids within the female genital tract.
  • the egg cells of all higher vertebrates are covered by a protective layer of nutrient cells, the so-called Corona radiata, and an inner mucopolysaccharide layer, the socalled Zona peilucida.
  • Lysosomal enzymes contained in the spermal acrosome enable the sperms to penetrate the protective layers and fuse with the egg cell. In this so-called acrosome reaction, sperms lose their foremost head portion of the outer plasma membrane but retain the rear portion of the plasma membrane with any substance adhering thereto until they have reached the cytoplasm of the egg ceil. For in vitro fertilization, the protective layers surrounding the egg cells are often removed by physicochemical means before sperms are added.
  • sperms may even enter into somatic cells (Brackett et al . , 1971: PNAS 68(2): 353).
  • a further use of the method according to the invention is thus possible, namely transfer of substances to somatic cells and transformed embryonal cells, e.g. with the object of conducting somatic gene therapy.
  • sperms need not necessarily undergo endocytosis with vesicles or granulae and, thus, with the substances to be transferred but may carry them "pick-a-back" on the spermal membrane into the egg cell or somatic cell where the transferred substances become effective.
  • each sperm becomes a vehicle for vesicles or granulae.
  • the number of transgenic descendants actually obtained therefore depends on the rate of inclusion (percentage of DNA-containing vesicles bound per sperm unit) and the rate of natural fertilization (successful pregnancy after insemination).
  • transgenic domestic animals such as cattle, sheep and the like
  • established animal breeding organizations may be involved.
  • the insemination centers where practically all sperm specimens for inseminating female animals and producing the entire descendant generation are obtained.
  • Another advantage of the method according to the invention is the elimination of many disturbing factors.
  • the selected gene may be introduced at one and the same time into all descendants of a population. For this purpose, one merely has to treat all spermal specimens with the gene-containing vesicles (or granulae). Presence of the transferred gene in the organisms of the descendant generation is easily verifiable (e.g. by taking blood samples) so that, thirdly, the generation interval is reduced as the animals may be used in breeding at a very early stage.
  • genes are available which should, of course, not be introduced at one and the same time; suitably, a single gene (or a small number of genes) per animal is introduced during the course of natural fertilization. This procedure is particularly preferred for experiments wherein individual (new) genes are tested for specific effects, efficiency or effects in various positions.
  • the method of the invention is ideally suited for today's breeding techniques.
  • the low transportation costs of embryos treated in accordance with the invention are of particular advantage.
  • pre-ovulatory eggs may be fertilized in vitro with substance-loaded sperm and, after successful development to the morula stage, subjected to deep-freezing or short-term preservation; they may then be shipped as transgenic embryos to any desired place at low cost.
  • Transgenic animals are required in research, too, for investigating specific questions.
  • the method of the invention is perfectly suited for laboratory practice as well as for studies on fertili z ation in vitro. 'Working in accordance with the method of the invention requires less time and personnel than any of the traditional methods of (embryo and) gene transfer.
  • a structural gene may be recovered as cDNA by means of RNA-dependent DNA polymerase (reverse transcriptase) from tissue-isolated messenger-RNA.
  • RNA-dependent DNA polymerase reverse transcriptase
  • the cDNA thus obtained permits identification and isolation of genomic DNA, i.e. the natural gene.
  • Recovery of genomic DNA fragments is state of the art (general description: Cooper, T.G. 1981: Biochemische Harvey-methoden, Walter de Gruyter, Berlin; Old, R.W., Pimrose, S.B., 1981: Principles of Gene Manipulation, Blackwell, Oxford; Maniatis et al., 1982: Molecular Cloning, Cold Spring Harbor).
  • a multitude of known DNA fragments from a variety of species are available at present in so-called gene banks (depositories). Also available are more than 4,000 genes of determined sequence derived from a variety of organisms, starting from SV 40-DNA up to the casein gene of cattle. The individual genes comprise up to several thousands of base pairs ( kb ) .
  • a chart of restriction enzymes is normally compiled, either empirically through all possible endonucleases or, if the nucleotide sequence is known, by tetra- or hexanucleotide comparison with any known restriction enzyme (data information by the European Molecular Biology Laboratory, Heidelberg).
  • Any structural gene may be linked with a promoter of choice (to improve gene expression). Promoter is called that part of a gene that induces transcription.
  • the structural gene of the growth hormone for instance, is positioned between two Bam Hi-recognition sites for restriction enzymes.
  • the Bam HI-fragment is isolated by digesting 100 ⁇ g DNA of the plasmide containing the gene for the growth hormone with 100 units of Bam HI (37oC, two hours). Digestion reaction terminated by adding a stopper solution (Maniatis et al ., 1982: Molecular Cloning, Cold Spring Harbor), the mixture being then applied to 0.7 % agarose gel.
  • a promoter metalothioneine commercially available in a plasmide pMMT 342 is isolated.
  • the metallothioneine promoter lies within an Eco RI - Bgl fragment. Once the fragment is present in pure form (see above), it is ligated with the structural gene (method: Maniatis et al., 1982: Molecular Cloning, Cold Spring Harbor). Ligation is possible with minute quantities. To obtain larger quantities of the new material set up in this manner, promoter and structural gene are incorporated (ligated) in a plasmide which, after transformation in bacteria, permits practically unlimited propagation.
  • a well known plasmide is pBR 322, the prototype of many presently used plasmides. It comprises various essential functional elements: "origin of replication (Ori)" and two genes coding for resistance to antibiotics (ampicillin, tetracvcline).
  • Such plasmides are known to have various sites where restriction enzymes may cut.
  • polylinkers which show a large number of recognition sites for restriction enzymes within a very short nucleotide sequence (50 bp), thus offering many possibilities of recombination.
  • Polylinker pSP 64 and, as a modification thereof, polylinker pSP 65 have been used successfully for such purposes .
  • Plasmides replicate extrachromosomally in bacteria, thus replicating the inserted DNA sequence, too, so that cloning of the desired gene to obtain larger quantities thereof (100 to 1,000 per batch) is possible.
  • the MT-structural gene fragment is ligated into polylinker pSP 65, the fragment being present in the form of Eco RI - Bgl II/Bam HI- Bam HI.
  • Polylinker pSP 65 also has one Eco RI- and one Bam HI- site; opening of these sites results in linearization of the plasmide. Two different cohesive ends corresponding to those of the fragment to be inserted have thus been created. Ligation produces two circular plasmides, namely the original plasmide through auto-ligation, and the desired plasmide having a size of 6.9 kb.
  • the gene is now present as part of a circular or linear plasmide. As such, it is dissolved in TE-buffer (0.01 M Tris, 0.001 M EDTA, 0.15 M PBS; pH 7.2) after having been purified in phenol/chloroform. For the subsequent steps of the method, concentration should be kept between 200 and 500 ⁇ g/ml, usually determined by photometer.
  • sperms carry complete genomes within their DNA-protecting membranes so that the DNA never comes into contact with degrading substances on the rather long way to the egg cells.
  • sperms became fairly reliable in taking the DNA compressed therein to the target cells.
  • any attempts at introducing additional genes into the sperms were bound to fail. Additional DNA attached to the outer surface of sperms must therefore be protected if it is to safely reach the egg cells.
  • Best suited for this purpose are artificially prepared vesicles in the form of liposomes. Liposomes are prepared from natural components of animal cells. Such components are commercially available in form of phospholipids (lecithin, phosphatidylethanolamine and the like).
  • lipid-soluble membrane fragments of erythrocytes For this purpose, blood of rabbits in native stage is treated with 0.15 % sodium heparin (about 50 ml per batch). The fresh blood is centrifuged at 3,000 rpm , the plasma being decanted. The procedure is repeated twice under addition of 0.15 M PBS to wash the erythrocytes, the leucocytes in the interphase being discarded.
  • the cells are then subjected to lysis in distilled water with 0.1 % Triton 100 over night to break open the cell membrane and dissolve the cell contents (haemoglobin).
  • lysis in distilled water with 0.1 % Triton 100 over night to break open the cell membrane and dissolve the cell contents (haemoglobin).
  • the membrane fragments are separated from the haemoglobin-containing supernatant.
  • the pellet is then taken up in a chloroform/methanol mixture (20 ml, 5:1) and mixed therewith over night under gentle agitation.
  • the liquid molecules are thus dissolved in chloroform while hydrophilic substances remain in the aqueous phase.
  • the mixture is then centrifuged at 3,000 rpm.
  • the upper phase contains water-soluble components, the interphase amphiphilic substances, and the lower (chloroform) phase the lipoid components, e.g. phosphatidylcholine, cholesterol, and glycolipids constituting the binding-specific epitopes of the prospective liposomes.
  • Concentration of the main components may be determined by high-pressure liquid chromatography (HPLC) but such determination is unneccessary when preparation is performed under standardized conditions.
  • lipid/chloroform solution 4 ml are used in the subsequent preparation of liposomes.
  • Required is a 25 ml round bottom flask wherein chloroform is evaporated at about 30oC by means of a rotary evaporator or an aspirator.
  • all components may be acquired commercially, a useful combination being e.g. phosphatidylcholine, cholesterol, phosphatidylserine and a glycolipid haying a terminal alpha-mannose group.
  • a useful combination being e.g. phosphatidylcholine, cholesterol, phosphatidylserine and a glycolipid haying a terminal alpha-mannose group.
  • the above components are mixed in a ratio of 5:1:3:2 and evaporated as described before.
  • Very stable and cell-compatible liposomes are obtained by using phosphatidylcholine and phosphatidylethanolamine in a ratio of 4:1. These compositions are particularly suited for the detergent method described below.
  • Membranes of sperms may be used according to a further embodiment.
  • Sperms freed of seminal plasma are treated under constant agitation with a chloroform/methanol mixture (5:1, v/v) to dissolve the lipid components of the membranes. Centrifuging at 3,000 rpm separates the chloroform phase containing the desired components from the other components. The purpose of this procedure is recovery of all required natural membrane components including the very important epitopes that permit specific binding.
  • Two phases are formed in this manner, a lower aqueous phase and an upper ether phase.
  • the two phases are emulsified on a vortex agitator (2 to 5 min., 30°C) until a fine emulsion is obtained.
  • Due to energetics, spherical uni- or multilamellar structures form of the lipoid substances while ether evaporates in part.
  • the vesicles thus forming enclose a small volume of the aqueous phase whereby dissolved DNA fragments are taken up in the interior of the liposomes .
  • the spherical form of the membrane components remains stable unless attacked by organic solvents. For that reason, residual ether must be removed as fast as possible from the emulsion. This is accomplished by immediate evaporation with the aid of a rotary evaporator or an aspirator under gentle shaking (20 min.).
  • a detergent sodium cholate
  • chloroform a detergent (sodium cholate) is admixed (20 ⁇ g at a phospholipid content of 20 to 50 ⁇ g ) before chloroform is evaporated.
  • a fatty layer composed e.g. of 50 ⁇ g lecithin (phosphatidylcholine), cholesterol, natural cell components and sodium cholate (ratio 5:1:2:3) remains on the inner surface of the flask.
  • lecithin phosphatidylcholine
  • sodium cholate ratio 5:1:2:3
  • the artificial vesicles thus obtained have surfaces similar to those of erythrocytes or sperms; in any case, their surfaces show epitopes that permit binding to sperms while their interior contains an aqueous phase comprising dissolved DNA.
  • liposomes are impermeable to macromolecular substances, the entrapped DNA is perfectly protected from degradation (e.g. DNases). Any other molecule maybe entrapped in liposomes in the same manner.
  • the liposomes are recovered by centrifugation at 40,000 g and taken up PBS.
  • the ratio pellet to solvent may be from 1:5 to 1:10.
  • PBS y be replaced with other buffer solutions as long as the pH is between 6.9 and 7.3 and a physiological concentration of 0.15 M is retained (with cells of mammals as target cells).
  • An example of suitable buffer solution is the so-called Ringer solution which has the following composition: 0.9 % NaCl (100 ml), 1.15 % KCl (4 ml), 2.1 % KH 2 PO 4 (1 ml), 3.82 % MgSO 4 7 H 2 O (1 ml) 1.3 % NaHCO 3 (2 ml, CO 2 -saturated).
  • Ringer solution which has the following composition: 0.9 % NaCl (100 ml), 1.15 % KCl (4 ml), 2.1 % KH 2 PO 4 (1 ml), 3.82 % MgSO 4 7 H 2 O (1 ml) 1.3 % NaHCO 3 (2 ml, CO 2 -saturated).
  • the DNA to be transferred (one or more functional groups of any type desired), i.e. several copies thereof in an aqueous solution (the number of copies depending on the concentration of DNA in the solution), is entrapped within a defined structure (vesicle or granula) that has surface-protruding structural elements (epitopes) capable of establishing close contact with certain binding molecules (lectins or antibodies).
  • a defined structure vesicle or granula
  • epitopes of liposomes are sterically identical or very similar to those on the surface of sperms; however, when unspecific binding forms ("coating") are employed, they may be of entirely different configuration or missing completely.
  • Sperms are recovered artificially by methods well described for domestic and laboratory animals (Paufler, S.K., 1974: Kunststoffliche Besamung I, Schaper, Hannover). Seminal plasma is removed from the ejaculate by centrifugation (400 rpm) and buffer solution (Ringer solution with 0.5 % fructose) is added. In practice, the collected sperm (several billions of sperms per ejaculate) is diluted and divided into portions; depending on the species, there may be obtained from 20 (rabbit) to 100 (bull) insemination specimen per ejaculate. It is important for the method of the invention that the diluent used is free of molecules which are capable of interacting with the respective binding molecules.
  • neuraminidase 1 mg/ml, Boehringer, Mannheim
  • a further possibility of binding liposomes to sperms is use of sperm-specific antibodies.
  • female animals rabbits
  • an ejaculate free of seminal plasma incomplete Freund's adjuvant
  • Second immunization takes place after two weeks and third immunization after an additional week.
  • blood (10 to 20 ml) is taken from the immunized animals, cooled and centrifuged at 3,000 rpm to recover the serum. Contained in the serum are (polyclonal) antibodies against epitopes on the spermal surface. Antisera thus obtained cause agglutination of rabbit blood even when diluted in a ratio of 1:1,000.
  • spermal antigen-specific antibodies permit two forms of binding between envelopes and sperms.
  • the envelope components are obtained from spermal membranes and the envelopes are prepared in the above described manner.
  • Bivalent or polyvalent antibodies then bind identical or similar structural elements on the surfaces of envelopes and sperms
  • unspecific adhesion of antibodies is utilized for covering the envelope surface with antibodies (coating).
  • Spermal antigen-specific binding sites of the antibodies will protrude from the enveloce surface and permit binding to sperms.
  • 0.5 ml of antiserum diluted in a ratio of 1 : 50 with Ringer solution (spermal agglutination titer at dilution of 1:1,000) are added to 0.5 ml of liposome suspension and incubated over night at 4°C.
  • Excess antibodies are washed (2 ml of Ringer solution) from the mixture by means of a fine-pore filter (millipores, 0.2 ⁇ m) and the purified liposomes on the filter are taken up in 0.5 ml of Ringer solution (counter-current flow).
  • the liposome suspension is centrifuged for 2 minutes at 12,000 rpm whereby large unsuited liposomes are precipitated in the pellet while liposomes of the desired size (200 to 800 ⁇ m) remain in the supernatant.
  • the liposomes are then bound to the surfaces of sperms. Liposomes and sperms are cautiously mixed at room temperature so that adsorption of the binding molecule-loaded liposomes on the specific epitopes of the sperm surface is completed within 20 minutes.
  • Ethidiumbromide-containing DNA was entrapped in liposomes and adsorbed on sperms.
  • Ethidiumbromide in aqueous solution maybe entrapped in liposomes, too, the liposomes then showing a pink glow under u.v. light.
  • Ethidiumbromide binds (intercalation) specifically to DNA which is then discernible in the liposomes by its green color. Adsorbed on sperms, it produces a green fringe around the sperms.
  • the surplus lectin binding sites (Con A)are best saturated just prior to insemination by adding 10 to 50 ⁇ l of alpha-mannose or alpha-glucose (0.01 M). The exact quantity of sugar to be added depends on the number of envelopes, the respective type of lectin and the sperms and may be determined for each system by simple preliminary tests.
  • the sperm specimen is ready for artificial insemination procedures.
  • LH gonadotropic hormone
  • HCG human chorion gonadotropin
  • a sperm specimen is deposited into the vagina with an insemination pipette.
  • the sperms migrate upwards through the female genital tract to arrive at the fertilization site (ampulla) where they enter the freshly ovulated eggs.
  • the gene copies come into the region of the amphimictic nucleus where the diploid chromosome set is formed.
  • the liposomes carried by sperms When the liposomes carried by sperms have reached the interior of the egg cell, they undergo lysis with cell-inherent enzymes. DNA thus released is capable of integrating in the genome. Successful integration into the embryo is verified by in situ hybridization.
  • the (transgenic) embryos (bicellular stage to blastocyst stage) are washed out of the fallopian tube or uterus of the rabbit (by surgical means in this particular case) and are then immobilized on an object slide by a fixing liquid (methanol/glacial acetic acid, 3:1, v/v).
  • pronase and colcemide cell cleavage inhibitor
  • pronase and colcemide cell cleavage inhibitor
  • phosphatidylcholine 8 mg
  • gangliosides 2 mg
  • sodium cholate 6 mg
  • Phosphatidylcholine and the gangliosides are dissolved in methanol and blended in the respective volumes in a glass flask.
  • Sodium cholate is weighed in as a powder.
  • methanol is evaporated by means of a rotary evaporator or an aspirator, An opalescent layer thus forms on the inner surface of the flask.
  • aqueous solution of substances is added to be transferred.
  • Concentration of pronase may be 100 ⁇ g/ml PBS (phosphate buffered saline), concentration of colcemide e.g. 50 ⁇ g/ml PBS.
  • the total amount of aqueous solution added is 1 ml. Due to the presence of a detergent, the lipid-containing layer on the inner surface of the flask dissolves completely. The mixture is then dialyzed against PBS for 5 hours under constant agitation (membrane permeable for molecules having a molecular weight below 10,000 d).
  • Liposomes spherical bodies containing the substances to be transferred are formed. Liposomes of varying size are obtained after dialysis, their average diameter being about 600 nm, i.e. below the visibility range of a light microscope; contained within these liposomes is a buffer solution of the substances to be transferred. (C) Surface treatment of liposomes.
  • neuraminidase As the membranes of the liposomes show terminal sialinic acid groups, 20 ⁇ g/ml of neuraminidase are added to the liposomes to remove the sialinic acids of the gangliosides by cleavage. Surface charge (negatively charged sialinic acid) of the liposomes is thus reduced, and sugar molecules originally hidden under sialinic acids are exposed to serve as additional binding sites for lectins such as Con A and PNA. After 30 minutes of incubation with neuraminidase (37°C), Con A and PNA (10 ⁇ g/ml each) are added to the suspension. The terminal sugar groups on the liposomes are thus saturated with lectins (incubation 15 min. at room temperature).
  • the sperms are advantageously loaded with size-calibrated liposomes as oversized liposomes are not suited for transport through the various cell barriers (Zona peilucida and membrane of egg cell) while undersized liposomes, due to their small lumen, cannot take up adequate volumes of substances.
  • the surface-treated liposomes are therefore filtered under moderate pressure (0.2 ⁇ m pore size of filter membrane). Undersized liposomes as well as free lectins and neuraminidase pass through the filter and are discarded. Rinsing the same filter from the reverse side results in a fraction comprising medium-sized as well as oversized liposomes. This fraction (about 1 ml) is centrifuged for 4 minutes at 12,000 g to pelletize oversized liposomes. Liposomes of the desired size remain in the supernatant.
  • Liposome-loaded sperm is now used for inseminating animals. After two days, the embryos are washed from the genital tract of the test animal and inspected under a microscope. Control embryos fertilized with sperm carrying "empty" liposomes show normal development, i.e. they reach a four-cell or eight-cell stage within a period of 48 hours after fertilization. Egg cells fertilized with sperms that have been loaded with pronase- or colcemide-containing liposomes are still in the zygote stage (i.e. a single cell which, in contrast to the unfertilized egg cell, shows two polar bodies, thus being identifiable under the microscope) after two days, some cells even showing phenomena of lysis.

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Abstract

Le procédé ci-décrit consiste à combiner des spermatozoïdes du type respectif, éventuellement modifiés par des moyens chimiques ou physiques, avec des vésicules ou des granules contenant des substances organiques ou inorganiques souhaitées et ensuite à mettre en contact les spermatozoïdes chargés avec les ovules ou les cellules somatiques dans des conditions intracorporelles ou extracorporelles. L'invention concerne également des compositions destinées à être utilisées dans ce procédé et des précurseurs desdites compositions. Pour finir, l'invention se rapporte aussi aux animaux transgéniques produits par le procédé.
EP87902077A 1986-03-03 1987-03-02 Procede pour transferer des substances organiques a des cellules-oeufs d'animaux, et compositions utilisees a cet effet Withdrawn EP0258427A1 (fr)

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DE3636991 1986-10-30
DE19863636991 DE3636991A1 (de) 1986-03-03 1986-10-30 Verfahren zur uebertragung organischer und/oder anorganischer substanzen auf ei- und/oder somazellen von tieren sowie entsprechende zusammensetzungen

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IT1228210B (it) * 1989-01-10 1991-06-05 Consiglio Nazionale Ricerche Procedimento per la introduzione di dna esogeno nelle cellule somatiche e germinali di animali.
US5614396A (en) * 1990-06-14 1997-03-25 Baylor College Of Medicine Methods for the genetic modification of endogenous genes in animal cells by homologous recombination
US5569824A (en) * 1991-01-04 1996-10-29 Baylor College Of Medicine Transgenic mice containing a disrupted p53 gene
DK0620849T3 (da) * 1992-01-07 2003-10-20 Elan Pharm Inc Transgene dyremodeller for Alzheimer's sygdom
AU2146092A (en) * 1992-05-28 1993-12-30 Scientific Dimensions Usa, Inc. Transgenic animal production with biolistically transformed spermatozoa
US5602307A (en) * 1992-08-12 1997-02-11 Baylor College Of Medicine Non-human animal having predefined allele of a cellular adhesion gene
WO1994005782A1 (fr) * 1992-09-10 1994-03-17 Trustees Of Tufts College Procede de production in vivo d'un organe transgenique par introduction du transgene dans une lumiere
US6717031B2 (en) 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease
US7312374B2 (en) 2001-09-18 2007-12-25 Avigenics, Inc Production of a transgenic avian by cytoplasmic injection
US7550650B2 (en) 2001-09-18 2009-06-23 Synageva Biopharma Corp. Production of a transgenic avian by cytoplasmic injection
US7135562B2 (en) 2002-03-14 2006-11-14 University Of Cincinnati Avian iFABP gene expression controlling region
US11519005B2 (en) 2017-09-28 2022-12-06 The Governors Of The University Of Alberta Retinoic acid-inducible gene I promoter and compositions and methods relating to same

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EP0081570A4 (fr) * 1981-06-12 1985-09-02 Univ Ohio Transformation genetique de zygotes.
NZ207393A (en) * 1983-08-05 1987-03-31 Neal Lloyd First Staining dna in living cells
EP0160692A1 (fr) * 1983-11-03 1985-11-13 DE WET, Johannes Martenis Jacob Procede de transfert de genes exogenes dans des plantes en utilisant le pollen comme vecteur

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