PRODUCTIONOFTRANSGENIGS BYGENETICTRANSFER INTOONE BLASTOMERE OfAN EMBRYO
BACKGROUND OF THE INVENTION
A transgenic animal is an animal which has integrated into its replicating complement of DNA of all or part of its cells one or more functional genes (that is, genes which occur in conjunction with controlling elements that allow for the expression of the gene) which are of exogenous origin. The transgene(s) of a transgenic animal have been artificially introduced into the animal at the gamete, zygote or early embryo stage. Such genes may have detectable effects, i.e., expression of an exogenous protein, for a limited period during development or throughout the life of the animal.
Animals can be completely transgenic; that is, all of the cells contain the transgene. Animals can also be partially transgenic, that is mosaic. Mosaic animals are those which result from the successful integration of introduced DNA into the DNA complement of only a portion of those cells of the animal. A mosaic may occur when, for example, a zygote is microinjected with DNA, but the DNA does not integrate into a chromosome until after one or more cell divisions occur. Mosaic animals have tissues made up of cells of different, mutually exclusive genotypes and/or phenotypes. Therefore, they may or may not be able to pass the transgene to their offspring through the germline cells.
Transgenic mammals have been made in which the animal produces an exogenous protein in milk, for example, tissue plasminogen factor expressed in goats, human anti- hemophilic factor IX in sheep, and human urokinase in the mouse. (See, for review article, Ebert, K.M. and J.P. Selgrath, "Changes in Domestic Livestock through Genetic Engineering" in Animal Applications in Mammalian Development, Cold Spring Harbor Laboratory Press, 1991.) Other transgenic animals (swine) have been produced which express porcine growth hormone under the control of promoter/enhancer elements originally isolated from Moloney murine leukemia virus or from cytomegalovirus (Ebert, K.M. et al . . Animal Biotechnology 1:145-159 (1990)). Transgenic mice have been made which express rat proenkephalin- chloramphenicol acetyltransferase fusion genes in tissues that normally express the proenkephalin gene (Zinn, S.A. et al . , J. Biol . Chem. 256:23850-23855 (1991). Transgenic animals are useful not only as improved agricultural stock or as a means of production for a protein, but have also been valuable tools for discovering the mechanisms by which gene expression is controlled, and are used as models for expression of recombinant genes. (See, for example, Low, M.J. et al . , Molecular Endocrinology 3:2028-2033 (1989)). Transgenic animals have been produced successfully by introducing foreign DNA into zygotes (see, for example, Wagner et al . , U.S. 4,873,191 (1989)) and into eggs (see, for example, Brinster, R.L. et al . , Proc. Natl Acad. Sci . USA 02:4438-4442 (1985)). However, methods of introducing DNA into zygotes or eggs suffer from a relatively low rate of success in producing a fully transgenic animal.
SUMMARY OF THE INVENTION
The invention described herein is a method to make a transgenic animal in which exogenous genetic material is introduced into at least one blastomere, that is, a totipotent cell of a multi-celled early embryo. All but one of the blastomeres are eliminated so as to leave one surviving blastomere which contains exogenous genetic material. The blastomere which is to live is placed in an environment which will provide appropriate conditions for the development of a transgenic animal. In the case of a mammal, the environment will allow for the development through embryonic and fetal development to term. In the case of other animals, the environment will allow for the development through embryonic development which normally occurs in the egg to hatching.
DETAILED DESCRIPTION
General Methods
The invention relates to methods of producing a transgenic animal by introducing one or more desired genes into at least one cell of an embryo in which all cells are totipotent, and selectively eliminating other cells of the embryo, such as by removal or by killing, so as to leave one surviving cell which contains, or will contain, depending on the order of the steps, an introduced gene or genes.
Cells of the early embryo are blastomeres. A blastomere is totipotent, meaning it has the capacity to develop into a complete, normal animal, given the appropriate conditions for development. Mammalian blastomeres are known to be totipotent up through the 8- cell stage of the embryo (See review article, Papaioannou, V.E. and K.M. Ebert, In: Experimental Approaches to Mammalian Embryonic Development, Pedersen, R. and J. Rossant, eds., 1986.)
The embryo to be genetically altered is preferably a two-cell embryo that results from the first cleavage of a zygote (fertilized ovum) . Two-cell embryos can be collected by flushing the oviduct of naturally mated or artificially inseminated females 1.5 to 2.5 days after insemination. Embryos can also be generated post- fertilization by using known methods to culture a zygote to the two-cell stage in culture medium in an incubator. In some animals, for example pigs, it may be necessary to collect recently fertilized eggs from the female following insemination in order to monitor cell division to the 2- cell stage in culture.
Any technique which allows for the addition of the exogenous genetic material to or into the genome can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. Preferably, the exogenous genetic material is introduced into the nucleus by microinjection. Microinjection of cells and more specifically, of nuclei, is known and is used in the art.
Embryos can be placed in culture medium and held in place by suction on a holding pipette. The nucleus of one blastomere is injected with a solution of DNA via an injection needle. Methods of microinjecting cells, such as ova or zygotes, are well known in the art, and are described, for example, in Brinster, R.L. et al. (Proc. Natl . Acad. Sci . USA 62:4438-4442 (1985)). The method generally requires a suitable liquid medium to sustain the cells to be manipulated and injected. Suitable media for the microinjection procedure in 2-cell mammalian embryos are, for instance, modified BMOC-2 containing HEPES salts, as described in Ebert, K.M. et al . (J. Embryol . Exp. Morph . 84:91-103 (1984)) or PBS medium supplemented with 20% fetal calf serum (Jura, J. et al . , Theriogenologγ 41:1259-1266 (1994)) or Brinster's medium plus 25 μM HEPES buffer (pH 7.4) (Brinster, R.L. et al . (Proc. Natl . Acad. Sci . USA 62:4438-4442 (1985)). A means to immobilize the cell to be injected may be necessary, such as a blunt pipet of a suitable diameter. The blunt pipet holds the cell by negative pressure. A sharp-tipped (1-2 μm, for example) injector pipet can serve as a means for introducing a solution of DNA into the nucleus of the cell. Swelling of the nucleus can be observed upon injection of the DNA. The means for holding and injecting the cells are controlled by appropriate instrumentation for micromanipulation. The process can be observed under a microscope using suitable magnification, e.g. 250x. DNA can be injected in a volume of 1-2 pi, optimally, or up to a maximum volume that does not cause irreparable damage to the nucleus or to the cell. Optimal concentrations of DNA to be used may need to be determined empirically, but integration frequencies have been good in fertilized eggs when several hundred gene copies are injected. (See, e.g., for fertilized ova from heifers, McEvoy, T.G. et al . (J. Reprod. Fert . Suppl . 43:297-289 (1991)). Concentrations of the DNA may vary. Preferably the concentration of DNA can vary from approximately 1 to 4 ng/μl. DNA to be injected may be genomic, cDNA, in linear or supercoiled form. The size of the DNA molecules injected is not critical to the success of the method, and can vary with the length of the gene or genes to be introduced. The usual size of the DNA should be long enough to include the desired functional gene to be transferred, and may range from a few kb to on the order of 10 kb. Artificial chromosomes constructed to introduce DNA into a blastomere can be considerably larger, up to hundreds of kb (Sun, T-Q. et al . , Nature Genetics 8:33-41 (1994)).
The non-injected blastomeres and any second, third, etc. injected blastomeres not intended to survive, are eliminated, preferably by killing them. Blastomeres not intended to become the surviving, altered blastomere of an embryo may be killed either before or after injection of the blastomere intended to become the surviving, altered blastomere. However, killing of the unwanted blastomere(s) is more conveniently done following the injection of the intended survivor. One method of killing is to intentionally rupture the nuclear and cytoplasmic membranes. Within 10 minutes of puncture, the majority of blastomeres lyse. Cellular debris fills the peri-vitelline space and indicates that the blastomere is non-viable. An alternative is to rupture the nuclear membrane of any non- injected blastomere(s) . This can be done by injecting the nucleus with a fluid (e.g., sterile culture medium) to expand the volume of the nucleus sufficiently to rupture the nuclear membrane and disperse the contents of the nucleus.
For animals whose embryonic stages do not naturally develop in utero, that is, egg-laying species such as fish, amphibians and insects, appropriate culturing in vitro can allow for the further development of the organism to a first stage that is independently motile and capable of feeding. This stage is a hatchling or newly hatched form in animals which naturally develop through their embryonic forms within eggs. For example, in animals such as fish, amphibians and insects, the appropriate culture medium and conditions can allow for the development of a blastomere to the stage normally occurring upon hatching of the egg, i.e. fry, tadpoles (for species of frogs) and larvae, respectively. Transgenic fish can be generated by methods similar to those described by Disney, J.E. et al . , in J". Exp. Zool . 248:335-344 (1988).
The 2-cell mammalian embryo treated in this manner can be either transferred to the oviduct of a recipient female shortly after lysis of the non-injected cell or can be cultured for a time and transferred to the oviduct or uterus, as desired to best match the developmental stage of the cultured embryo to the stage of pregnancy or pseudopregnancy of the host female. As previously shown (here, in experiments on mice; Papaioannou, V.E. and Ebert, K.M. Developmental Dynamics 203:393-398 (1995)) the surviving blastomere can give rise to a viable fetus and neonate.
In vitro incubation for some period of time is possible before transferring the embryo into an appropriate female animal to serve as mother. Preferably, the surviving blastomere of the embryo is transferred to an appropriate host animal with as little delay as possible. The animal which serves as surrogate mother for the development of the manipulated blastomere can be any animal that can provide the appropriate hormonal and nutritional environment for the growth and development of the embryo to term. Such an animal can be a female which is pregnant with embryos at or close to the same stage of development as the altered embryo or embryos which are transferred to her uterus. Alternatively, the host female can be induced by appropriate treatment to a pseudopregnant state. Preferably, the surrogate mother animal is of the same species as the embryo. Usually, this is required for the development of the embryo to term. However, some exceptions are known. For example, one species of antelope can in some cases serve as surrogate mother for gestation of an embryo of a different species of antelope.
In some cases, it may be advantageous to culture in vi tro the surviving blastomere in which genetic material has been introduced, for a period of time which will allow for testing of the presence of the introduced exogenous genetic material. For instance, one cell of a 4-cell stage embryo may be removed and its DNA tested for the presence of the exogenous genetic material introduced into one blastomere at the 2-cell stage. PCR or other methods suitable to the analysis of small amounts of DNA may be used to test for the presence or absence of a particular DNA sequence in a sample of an embryo while it is still in a stage of development when all cells are totipotent. A decision to transfer the embryo to a female for further development may be made, depending on whether the exogenous genetic material is detected in the sample cell of the embryo.
As with the injection of zygotes and eggs, it is expected that there will be several possible outcomes from this process of introducing DNA to one or more blastomeres and killing blastomeres to produce one altered survivor. If the microinjected DNA is integrated successfully into a replicating genetic unit of the host blastomere, such as a chromosome, the result is that the surviving microinjected blastomere, given the proper conditions, will grow and divide through the normal stages of development to form a transgenic animal. A second possible result is that the microinjected DNA is never integrated into nuclear genetic material of the blastomere, so that the introduced gene or genes are not expressed in the descendants of the microinjected cell, i.e., the animal that develops from the blastomere. A third possible result is that the introduced genetic material is not incorporated into the genome of the blastomere before the first DNA replication after the introduction occurs, but is incorporated into the genome some time after DNA replication occurs. The result is a mosaic animal.
It is preferable to introduce the exogenous genetic material into a blastomere at the earliest possible time in the 2-cell stage of the embryo so as to allow the maximum time for the incorporation of the exogenous genetic material into the genome before DNA replication occurs. This should increase the probability that the animal that results from the growth and development of the blastomere is a uniformly transgenic animal, rather than a mosaic of cells of different genotypes.
For insects, the integration of exogenous DNA may require the use of a transformation vector comprising the terminal repeats of a transposable element such as the P element of Drosophila, along with a helper vector which can supply a compatible functional transposase (See review article by J. Crampton et al . , Parasitologγ Today 6:31-36 (1990) .) The Drosophila melanogaster P element transposon has been used successfully as a transformation vector in this and in other species of Drosophila with the retention of normal expression of the introduced genes (Cooley, L. et al . . Trends Genet. 46:254-258 (1988)), and may have wider application in other insects. The probability of producing a uniformly transgenic animal, rather than a mosaic animal, should also be increased over the method of injecting a zygote or ovum with DNA, because the blastomere has the full diploid complement of DNA in one nucleus, twice the amount of DNA which is the target in the zygote, either the male or female pronucleus. This, in effect, presents twice the number of target sequences for the integration of the exogenous DNA in a blastomere compared to a zygote.
As an alternative to introducing genetic material which does not carry a functional origin of replication and therefore must rely on integration into a chromosome which has such an origin, an artificial chromosome carrying a desired gene may be introduced (See, for example of human artificial episomal chromosome, Sun, T-Q. et al . in Nature Genetics 8:33-41 (1994)).
To test for the success of the method, cell or tissue samples may be taken from the animal at a selected stage of development. Depending on the animal, the embryo, fetus, neonate, developing offspring or adult can be tested for the presence of the transgene by well-known techniques in molecular biology. Such techniques involve, for example, obtaining a sample of DNA from the fetus, neonate or developing offspring and, for example, testing it by Southern hybridization using a probe specific for the transgene, or by using primers for the specific amplification of the transgene by PCR, or by other suitable methods.
Germ-line transmission can be tested through either the analyses of the DNA of germ cells, as can be done for somatic cells, as above. Populations of germ cells, like populations of somatic cells, can be mosaic. Therefore, breeding experiments may also be done to distinguish uniformly transgenic animals from mosaic animals.
Applications and Further Considerations The method of making transgenic animals can be applied to a wide range of animals, including, for example, amphibians, fish and mammals, including primates. In some classes or subclasses of animals, for example in birds, the 2-cell stage of the embryo is not easily accessible to isolation to allow for this type of manipulation.
The present invention has application in the genetic transformation of multicellular eukaryotic animals which have in their reproductive cycle an early embryonic form characterized by a plurality of totipotent cells, such as a blastomere stage. Examples of such organisms include amphibians, reptiles, birds, mammals, bony fishes, cartilaginous fishes, cyclostomes, arthropods, insects, mollusks and thallophytes. Preferred organisms are those whose early embryos are amenable to isolation and micromanipulation following natural, artificial or in vi tro fertilization of ova, or whose zygotes can be isolated and cultured to a 2-cell embryo.
The invention is particularly useful in the breeding of animals, especially ones of agricultural value, to obtain species having a genetic makeup which results in an animal having more desirable characteristics. The source of the exogenous genetic material can be from animals or plants, viruses, bacteria or protozoa, for instance. The exogenous genetic material can be synthetic equivalents of naturally occurring genetic material or totally new synthetically produced genetic material. The exogenous genetic material can be from the same species as the blastomere being transfected, or from a different species. Thus, the invention can be used to modify a species. Modification of a species can be obtained when the new genotype including the exogenous genetic material occurs in the germline cells of the animal that develops from the altered blastomere. In some cases, it may be advantageous to use the transgenic embryo as a system to test for the timing and extent of expression of certain genes during the development of the animal. This can be done using known methods in the art. One method is, for example, recovering an embryonic or fetal animal, isolating polyA* RNA from one or more tissue types of interest, and testing the polyA* RNA for the presence of sequences homologous to the transgene.
The number of copies of the DNA sequences which are added to a blastomere is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the incorporation of the exogenous genetic material to occur in such a way that the exogenous gene(s) and/or controlling sequences(s) can be expressed. Theoretically, only one copy is required. However, it is preferable that numerous copies are introduced, for example, 100-1,000 copies of a gene, in order to insure that at least one copy is functional. A successful result of injecting a zygote of a mouse with multiple copies of linear DNA has been, typically, a transgenic mouse with all or some of its cells containing a tandem array of copies of the injected gene integrated at a single random site in one of its chromosomes (Palmiter, R.D. and R.L. Brinster Ann. Rev. Genet . 20:465-499 (1986)). Generally, there is no advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences unless an enhanced phenotypic expression of the exogenous DNA sequences is desired. There are instances where more than one functional copy of the exogenous DNA sequences may not be desirable, for example when the exogenous DNA sequences work in conjunction with endogenous DNA sequences of the organism to produce a particular product.
The physical effects of the alteration in the genome of the altered blastomere must not be so great as to physically destroy the viability of the blastomere. The biological limit of the number and variety of DNA sequences will vary depending upon the particular blastomere and the functions of the exogenous genetic material. The genetic material, including the exogenous genetic material, of the resulting blastomere must be biologically capable of initiating and maintaining the differentiation and development of the blastomere into a functional organism. From a biological consideration, relating to mitosis, when the exogenous genetic material is a chromosome, the amount inserted will not be greater than one chromosome and preferably will be less than one chromosome.
The particular composition or form of the exogenous genetic material is not critical. If a particular trait is desired to be incorporated into an organism, then the genetic material needs to contain the DNA sequence or sequences, or gene or loci, which code for the trait. Thus, whether the exogenous genetic material is a whole chromosome, a portion of a chromosome, purified or unpurified native DNA or synthetic or semi-synthetic DNA, or chromosomal complexes, is dependent upon the particular trait or traits sought to be incorporated into the zygote and ease of obtaining that trait. Techniques for obtaining segments of DNA by gene excising, splicing, synthesis, isolation, purification, cloning and the like are known in the art. Enzymes used in such processes, vectors and hosts for cloning of recombinant DNA, screening and selection of cloned DNA and detection and analysis of expression of cloned genes are also known in the art. (See, e.g., Ausubel, F.M. et al . , eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., (1996)).
Alterations in the phenotype can be the expression of one or more genes which are not naturally occurring in the host species, such as genes naturally occurring in a different species. Alternatively, the gene or genes introduced can be altered or mutated forms of natural genes, or genes which have been designed to express a designed product. Alteration of the expression of a phenotype also includes an enhancement or diminution in the expression of a phenotype or an alteration in the promotion and/or control of a phenotype including the addition of a new promoter and/or controlling element or supplementation of an existing promoter and/or controlling element of a phenotype.
Depending upon the particular trait or traits which are desired in the animal, it may be necessary to include in the introduced DNA the controlling elements responsible for expression of the gene(s) coding for the gene product causing the trait, particularly if the genotype of the blastomere contains no similar gene whose control region could be used to regulate the gene or if greater activity is desired. In some cases, it may be desirable to introduce back into the same species a gene originally isolated from that species, which has been altered to produce a different gene product from that produced in unaltered animals. In other cases, it may be desirable to introduce back into the same species a gene originally isolated from that species which has been put under the control of controlling elements isolated from a different organism or from a virus. In some instances it may be desirable to include a control region, e.g., an artificial one spliced to the exogenous gene, which will activate the gene when the organism is exposed to a different stimulus other than the natural stimulus of the gene. Definitions
"Genetic material" comprises DNA either purified or in a native state such as a fragment of a chromosome or a whole chromosome, either naturally occurring, synthetically prepared or partially synthetically prepared, DNA which constitutes a gene or genes, and gene chimeras, e.g., created by ligation of different DNA sequences. Genetic material may include DNA sequences from a plasmid, virus or phage. "Embryo" is the earliest multicelled phase in the development of a multicelled animal, having at least 2 cells, following the zygote phase.
"Blastomere" is a cell of any of the early embryo stages occurring after the cleavage of the zygote, through the 8-16 cell morula stage.
"Exogenous genetic material" is genetic material not obtained from or not naturally forming a part of the particular blastomere which is to be genetically altered. "Gene" is the smallest, independently functional unit of genetic material which codes for a protein or RNA product and comprises at least one nucleic acid sequence. A gene can include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
"Controlling elements" for genes include promoter regions, ribosome binding sequences, terminators, enhancers and the like. They are nucleic acids having sequences known in molecular biology to determine the timing and extent of expression of genes and can act at the DNA level (e.g., promoters) or at the RNA level (e.g., ribosome binding sequences) , often in conjunction with other molecules such as regulatory proteins.
"Genotype" is the genetic constitution of an organism. "Phenotype" is a collection of morphological, physiological and biochemical traits possessed by a cell or organism that results from the interaction of the genotype and its environment. "Chromosome" is a discrete replicating unit of the genome carrying many genes. Each eukaryotic chromosome consists of a very long molecule of duplex DNA and an approximately equal mass of proteins. It is visible as a morphological entity only during the action of cell division.
"Cell cycle" is the period from one cell division to the next.
"Genome" is the full complement of genetic material which is normally replicated from one generation of a cell to the next, and which determines the genotype of a cell.
"Transfection" of eukaryotic cells is the acquisition of new genetic markers by incorporation of added DNA.
"Zygote" is a fertilized egg produced by fusion of two gametes. "Totipotent" means capable of giving rise to a complete organism by division and cell specialization.