WO1991019796A1 - Procede de recombinaison homologue dans des cellules animales et vegetales - Google Patents

Procede de recombinaison homologue dans des cellules animales et vegetales Download PDF

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WO1991019796A1
WO1991019796A1 PCT/US1991/004006 US9104006W WO9119796A1 WO 1991019796 A1 WO1991019796 A1 WO 1991019796A1 US 9104006 W US9104006 W US 9104006W WO 9119796 A1 WO9119796 A1 WO 9119796A1
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cell
gene sequence
animal
gene
cells
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PCT/US1991/004006
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English (en)
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Allan Bradley
Ann C. Davis
Paul Hasty
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Baylor College Of Medicine
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Priority to AU81823/91A priority Critical patent/AU654284B2/en
Priority to JP91511760A priority patent/JPH05507853A/ja
Publication of WO1991019796A1 publication Critical patent/WO1991019796A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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 is directed toward recombinant DNA
  • the invention further pertains to the
  • animals have been engineered to contain gene sequences that are not normally or naturally present in an unaltered animal.
  • the techniques have also been used to produce animals which exhibit altered expression of naturally present gene sequences.
  • the animals produced through the use of these methods are known as either "chimeric” or "transgenic” animals.
  • a chimeric animal only some of the animal's cells contain and express the introduced gene sequence, whereas other cells have been unaltered.
  • the capacity of a chimeric animal to transmit the introduced gene sequence to its progeny depends upon whether the introduced gene sequences are present in the germ cells of the animal. Thus, only certain chimeric animals can pass along the desired gene sequence to their progeny. In contrast, all of the cells of a "transgenic" animal contain the introduced gene sequence. Consequently, a transgenic animal is capable of transmitting the introduced gene sequence to its progeny.
  • the most widely used method through which transgenic animals have been produced involves injecting a DNA molecule into the male pronucleus of a fertilized egg (Brinster, R.L. et al.. Cell .22:223 (1981); Costantini, F. et al.. Nature 294:92 (1981); Harbers, K. et al.. Nature 293:540 (1981); Wagner, E.F. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 78:5016 (1981) ; Gordon, J.W. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 72:1260 (1976)).
  • the gene sequence being introduced need not be incor- porated into any kind of self-replicating plasmid or virus (Jaenisch, R., Science, 240:1468-1474 (1988)). Indeed, the presence of vector DNA has been found, in many cases, to be undesirable (Hammer, R.E. et al.. Science 235:53 (1987) ; Chada, K. et al.. Nature 319:685 (1986); Kollias, G. et al.. Cell 4.6:89 (1986); Shani, M. , Molec. Cell. Biol. 6.:2624 (1986); Chada, K. et al.. Nature 314:377 (1985); Townes, T. et al..
  • the injected DNA molecules may be incorporated at several sites within the chromosomes of the fertilized egg, in most instances, only a single site of insertion is observed (Jaenisch, R. , Science. 240:1468-1474 (1988); Meade, H. et al. (U.S. Patent 4,873,316)).
  • the DNA molecule Once the DNA molecule has been injected into the fertilized egg cell, the cell is implanted into the uterus of a recipient female, and allowed to develop into an animal. Since all of the animal's cells are derived from the implanted fertilized egg, all of the cells of the resulting animal (including the germ line cells) shall contain the introduced gene sequence.
  • the resulting animal will be a chimeric animal.
  • breeding and inbreeding such animals it has been possible to produce heterozygous and homozygous transgenic animals. Despite any unpredictability in the formation of such transgenic animals, the animals have generally been found to be stable, and to be capable of producing offspring which retain and express the introduced gene sequence.
  • microinjection causes the injected DNA to be incorporated into the genome of the fertilized egg through a process involving the disruption and alteration of the nucleotide sequence in the chromosome of the egg at the insertion site, it has been observed to result in the alteration, disruption, or loss of function of the endogenous egg gene in which the injected DNA is inserted. Moreover, substantial alterations (deletions, duplications, rearrangements, and translocations) of the endogenous egg sequences flanking the inserted DNA have been observed (Mahon, K.A. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 85:1165 (1988); Covarrubias, Y. et al.
  • mice FASEB J. 2:117 (1989); Jaenisch, R. , Science 240:1468-1474 (1988)).
  • the success rate for producing transgenic animals is greatest in mice. Approximately 25% of fertilized mouse eggs into which DNA has been injected, and which have been implanted in a female, will become transgenic mice. A lower rate has been thus far achieved with rabbits, sheep, cattle, and pigs (Jaenisch, R. , Science 240:1468-1474 (1988); Hammer, R.E. et al.. J. Animal. Sci. 63:269 (1986); Hammer, R.E. et al.. Nature 315:680 (1985); Wagner, T.E. et al..
  • the lower rate may reflect greater familiarity with the mouse as a genetic system, or may reflect the difficulty of visualizing the male pronucleus of the fertilized eggs of many farm animals (Wagner, T.E. et al.. Therio ⁇ enolo ⁇ y 21:29 (1984)).
  • the production of transgenic animals by microinjection of DNA suffers from at least two major drawbacks. First, it can be accomplished only during the single-cell stage of an animal's life. Second, it requires the disruption of the natural sequence of the DNA, and thus is often mutagenic or teratogenic (Gridley, T. et al.. Trends Genet. 3:162 (1987)).
  • Chimeric and transgenic animals may also be produced using recombinant viral or retroviral techniques in which the gene sequence is introduced into an animal at a multi- cell stage.
  • the desired gene sequence is introduced into a virus or retrovirus.
  • Cells which are infected with the virus acquire the introduced gene sequence. If the virus or retrovirus infects every cell of the animal, then the method results in the production of a transgenic animal. If, however, the virus infects only some of the animal's cells, then a chimeric animal is produced.
  • the general advantage of viral or retroviral methods of producing transgenic animals over those methods which involve the microinjection of non-replicating DNA, is that it is not necessary to perform the genetic manipulations at a single cell stage.
  • retroviral methods for producing chimeric or transgenic animals have the advantage that retroviruses integrate into a host's genome in a precise manner, resulting generally in the presence of only a single integrated retrovirus (although multiple insertions may occur) .
  • Rearrangements of the host chromosome at the site of integration are, in general, limited to minor deletions (Jaenisch, R. , Science J24J):1468-1474 (1988); see also, Var us, H. , In: RNA Tumor Viruses (Weiss. R. et al.. Eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp.
  • Chimeric animals have, for example, been produced by incorporating a desired gene sequence into a virus (such as bovine papilloma virus or polyoma) which is capable of infecting the cells of a host animal. Upon infection, the virus can be maintained in an infected cell as an extrachromosomal episome (Elbrecht, A. et al. , Molec. Cell. Biol. 2:1276 (1987); Lacey, M. et al.. Nature 322:609 (1986); Leopold, P. et al.. Cell 51:885 (1987)). Although this method decreases the mutagenic nature of chimeric/transgenic animal formation, it does so by decreasing germ line stability, and increasing oncogenicity. -1-
  • a virus such as bovine papilloma virus or polyoma
  • Pluripotent embryonic stem cells are cells which may be obtained from embryos until the early post-implantation stage of embryogenesis. The cells may be propagated in culture, and are able to differentiate either in vitro or in vivo upon implantation into a mouse as a tumor. ES cells have a normal karyotype (Evans, M.J. et al.. Nature 292:154-156 (1981); Martin, G.R. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 78:7634-7638 (1981)).
  • ES cells Upon injection into a blastocyst of a developing embryo, ES cells will proliferate and differentiate, thus resulting in the production of a chimeric animal. ES cells are capable of colonizing both the somatic and germ-line lineages of such a chimeric animal (Robertson, E. et al.. Cold Spring Harb. Conf. Cell Prolif. 10:647-663 (1983); Bradley A. et al.. Nature 309:255-256 (1984); Bradley, A. et al.. Curr. TOP. Devel. Biol. 20:357-371 (1986); Wagner, E.F. et al.. Cold Sprin ⁇ Harb. S ⁇ p. Quant. Biol.
  • ES cells are cultured in vitro. and infected with a viral or retroviral vector containing the gene sequence of interest.
  • Chimeric animals generated with retroviral vectors have been found to have germ cells which either lack the introduced gene sequence, or contain the introduced sequence but lack the capacity to produce progeny cells capable of expressing the introduced sequence (Evans, M.J. et al.. Cold Spring Harb. Symp. Quant. Biol. 50:685-689 (1985); Stewart, CL. et al.. EMBO J. 4:3701-3709 (1985); Robertson, L. et al..
  • ES cells may be propagated in vitro. It is possible to manipulate such cells using the techniques of somatic cell genetics. Thus, it is possible to select ES cells which carry mutations (such as in the hprt gene (encoding hypoxanthine phosphoribosyl transferase) (Hooper, M. et al.. Nature 326:292-295 (1987); Kuehn, M.R. et al.. Nature 326:295-298 (1987)) .
  • hprt gene encoding hypoxanthine phosphoribosyl transferase
  • Such selected cells can then be used to produce chimeric or transgenic mice which fail to express an active HPRT enzyme, and thus provide animal models for diseases (such as the Lesch-Nyhan syndrome which is characterized by an HPRT deficiency) (Doetschman, T. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 85:8583-8587 (1988)) .
  • diseases such as the Lesch-Nyhan syndrome which is characterized by an HPRT deficiency
  • U.S.A. Proc. Natl. Acad. Sci. 85:8583-8587 (1988)
  • the above-described methods permit one to screen for the desired genetic alteration prior to introducing the trans- fected ES cells into the blastocyst.
  • One drawback of these methods is the inability to control the site or nature of
  • hprt-deficient Chinese hamster ovary (CHO) cells have been transformed with the CHO hprt gene in order to produce a prototrophic cell line (Graf, L.H. et al.. Somat. Cell Genet. 5:1031-1044 (1979)).
  • Foloer et al. examined the fate of a thymidine kinase gene (tk gene) which had been microinjected into the nuclei of cultured mammalian cells. Recipient cells were found to contain from 1 to 100 copies of the introduced gene sequence integrated as concatemers at one or a few sites in the cellular genome (Folger, K.R. et al.. Molec.
  • the cauliflower mosaic virus (Brisson, N. et al.. Nature 310:511-514 (1984) has been particularly useful for this purpose (Shah, D.M. et al.. Science 233:478-481 (1986); Shewmaker, C.K. et al.. Virol. 140:281-288 (1985).
  • Vectors have also been prepared from derivatives of RNA viruses (French, R. et al.. Science 231:1294-1297 (1986). Techniques of microinjection (Crossway, A. et al., Molec. Gen. Genet. 202:179-185 (1986); Potrykus, I. et al.. Molec. Gen. Genet.
  • a major deficiency of present methods for gene manipulation in plants is the difficulty of selecting the desired recombinant cell (Brunold, C. et al.. Molec. Gen. Genet. 208:469-473 (1987)).
  • kanamycin resistance and nitrate reductase deficiency have been used as selectable markers (Brunold, C. et al.. Molec. Gen. Genet. 208:469-473 (1987)).
  • Figure l illustrates the use of replacement vectors and insertion vectors in gene targeting.
  • Figure 1A is a diagrammatical representation of the use of a replacement vector in gene targeting;
  • Figure IB illustrates the use of an insertion vector to produce subtle mutations in a desired gene sequence.
  • Figure 2 is a diagrammatical representation of a DNA molecule which has a region of heterology located at a proposed insertion site.
  • Figure 2A shows a construct with a 2 kb region of heterology.
  • Figure 2B shows a construct with a 26 base long region of heterology which has been linearized at the center of the region of heterology.
  • Figure 2C shows a construct with a region of heterology located internal to the region of homology at which recombination is desired.
  • Figure 2C the normal BamHI site of the vector has been changed to an Nhel site and the normal EcoRI site of the vector has been changed to a BamHI site.
  • the vector is> linearized with Xhol.
  • Figure 3 is a diagrammatical representation of the mechanism through which a "humanized" gene may be introduced into a chromosomal gene sequence in a one step method.
  • Figure 4 is a diagrammatical representation of the mechanism through which a large gene may be introduced into a chromosomal gene sequence so as to place the gene under the transcriptional control of a heterologous promoter (for example, to place a human gene under the control of a mouse gene) .
  • the first step is additive and the second is a replacement event.
  • Figure 4A shows the first step of the process
  • Figure 4B shows the second step of the process.
  • the repair recombination event may be configured to remove all of the mouse coding exons if desired.
  • Figure 5 is a diagramatical representation of the use of a positive selection/ negative selection "cassette" to introduce subtle mutations into a chromosome.
  • Figure 6 is a diagrammatical representation of a multi- step method ( Figures 6A-6E) for introducing small or large desired gene sequences into a contiguous region of a cell's genome. The figure illustrates a vector capable of facilitating the sequential addition of overlapping clones to construct a large locus. Every step is selectable.
  • FIG. 7 is a diagrammatical representation of the vectors used in a co-electroporation experiment to mutate the hprt gene.
  • Figure 8 illustrates the predicted structure of the hprt gene following homologous recombination of the IV6.8 vector.
  • HR is the predicted size fragment indicative of the homologous recombination event.
  • End, D is the endogenous fragment, duplicated by the recombination event.
  • Figure 9 shows the reversion of homologous recombinants generated with insertion vectors.
  • Figure 10 illustrates the use of Poly A selection as a means for selecting homologous recombination events.
  • Figure 11 illustrates the use of the invention to introduce insertions into the sequence of a desired gene of a cell.
  • Figure 11B illustrates the src 14 vector used to introduce mutations into the c-src locus
  • Figure 11C illustrates the subtle mutation introduced through the use of this vector.
  • Figure 12 illustrates the use of the invention to introduce substitutions into the sequence of a desired gene of a cell.
  • Figure 12B illustrates the src 33 vector used to introduce mutations into the c-src locus;
  • Figure 12C illustrates the subtle mutation introduced through the use of this vector.
  • Figure 13 illustrates a comparison between targeted and random recombinational events.
  • a random recombinational event although concatemers can excise duplications, one copy of the vector must remain in the genome.
  • a targeted recombinational event all sequences, except the desired sequence is excised from the genome.
  • the present invention provides a method for obtaining a desired animal or non-fungal plant cell which contains a predefined, specific and desired alteration in its genome.
  • the invention further pertains to the non-human animals and plants which may be produced from such cells.
  • the invention additionally pertains to the use of such non-human animals and plants, and their progeny in research, medicine, and agriculture.
  • the invention provides a method for obtaining a desired animal or non-fungal plant cell which contains a desired non-selectable gene sequence inserted within a predetermined gene sequence of the cell's genome, which method comprises: A.
  • the invention further includes the embodiments of the above-described method wherein the DNA molecule contains a detectable marker gene sequence, and/or wherein the DNA molecule is introduced into the precursor cell by subjecting the precursor cell and the DNA molecule to electroporation (especially wherein in step B, the precursor cell is simultaneously subjected to electroporation with a second DNA molecule, the second DNA molecule containing a detectable marker gene sequence) .
  • the invention further includes the embodiments of the above-described method wherein the desired cell is a non- fungal plant cell, a somatic animal cell (especially one selected from the group consisting of a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, a non-human primate and a human) , a pluripotent animal cell (especially one selected from the group consisting of a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and a non-human primate) .
  • a somatic animal cell especially one selected from the group consisting of a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and a non-human primate
  • the invention includes with the embodiment wherein the pluripotent cell is an embryonic stem cell.
  • the invention also includes the embodiments of the above-described methods wherein the desired gene sequence is substantially homologous to the predetermined gene sequence of the precursor cell and/or wherein the desired gene sequence is an analog (and especially a human analog) of the predetermined sequence of the precursor cell.
  • the invention also includes the embodiment wherein the desired gene sequence encodes a protein selected from the group consisting of: a hormone, an immunoglobulin, a receptor molecule, a ligand of a receptor molecule, and an enzyme.
  • the invention also includes a non-fungal plant cell which contains an introduced recombinant DNA molecule containing a desired gene sequence, the desired gene sequence being flanked by regions of homology which are sufficient to permit the desired gene sequence to undergo homologous recombination with a predetermined gene sequence of the genome of the cell.
  • the invention also includes a non-human animal cell which contains an introduced recombinant DNA molecule containing a desired gene sequence, the desired gene sequence being flanked by regions of homology which are sufficient to permit the desired gene sequence to undergo homologous recombination with a predetermined gene sequence of the genome of the cell.
  • the invention also includes the desired cell produced by any of the above-described methods.
  • the invention also includes a non-human animal containing a cell derived from the above-described desired cell, or a descendant thereof, wherein the animal is either a chimeric or a transgenic animal, and particularly includes the embodiment wherein the non-human animal and the desired cell are of the same species, and wherein the species is selected from the group consisting of: a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and a non-human primate.
  • the invention also includes a non-fungal plant containing a cell derived from the above-described desired non-fungal plant cell, wherein said non-fungal plant is either a chimeric or a transgenic plant.
  • the invention also includes a method of gene therapy which comprises introducing to a recipient in need of such therapy, a desired non-selectable gene sequence, the method comprising: A. providing to the recipient an effective amount of a DNA molecule containing the desired non-selectable gene sequence, wherein the DNA molecule additionally contains two regions of homology which flank the desired gene sequence, and which are sufficient to permit the desired gene sequence to undergo homologous recombination with a predetermined gene sequence present in a precursor cell of the recipient; B.
  • the invention includes the embodiments of the above-stated method wherein the recipient is a non- fungal plant, or a human or a non-human animal (particularly a non-human animal is selected from the group consisting of: a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, a non-human primate and a human) .
  • the invention also provides a method for obtaining a desired animal or non-fungal plant cell which contains a desired non-selectable gene sequence inserted within a predetermined gene sequence of the cell's genome, which method comprises: A.
  • the invention also includes the embodiment wherein the cell is deficient in an HPRT, APRT, or TK enzyme, and wherein the selectable gene sequence expresses an active HPRT, APRT, or TK enzyme, and wherein the first set of selective culture conditions comprises incubation of the cell under conditions in which the presence of an active HPRT, APRT, or TK enzyme in the cell is required for growth, and wherein the second set of selective culture conditions comprises incubation of the cell under conditions in which the absence of an active HPRT, APRT, or TK enzyme in the cell is required for growth.
  • the present invention concerns a method for introducing DNA into the genome of a recipient plant or animal cell.
  • the method may be used to introduce such DNA into germ line cells of animals (especially, rodents (i.e. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle and non-human primates) in order to produce chimeric or transgenic animals.
  • the methods may also be used to introduce DNA into plant cells which can then be manipulated in order to produce chimeric or transgenic plants.
  • the method may be used to alter the somatic cells of an animal (including humans) or a plant.
  • the plants and plant cells which may be manipulated through application of the disclosed method include all multicellular, higher (i.e. non-fungal or non-yeast) plants.
  • the present invention provides a method for introducing a desired gene sequence into a plant or animal cell.
  • it is capable of producing chimeric or transgenic plants and animals having defined, and specific, gene alterations.
  • An understanding of the process of homologous recombination (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), which reference is incorporated herein by reference) is desirable in order to fully appreciate the present invention.
  • homologous recombination is a well-studied natural cellular process which results in the scission of two nucleic acid molecules having identical or substantially similar sequences (i.e.
  • homologous a region of DNA is intended to generally refer to any nucleic acid molecule.
  • the region may be of any length from a single base to a substantial fragment of a chromosome.
  • the molecules For homologous recombination to occur between two DNA molecules, the molecules must possess a "region of homology" with respect to one another. Such a region of homology must be at least two base pairs long. Two DNA molecules possess such a "region of homology" when one contains a region whose sequence is so similar to a region in the second molecule that homologous recombination can occur. Recombination is catalyzed by enzymes which are naturally present in both prokaryotic and eukaryotic cells. The transfer of a region of DNA may be envisioned as occurring through a multi-step process. If either of the two participant molecules is a circular molecule, then the above recombination event results in the integration of the circular molecule into the other participant.
  • a particular region is flanked by regions of homology (which may be the same, but are preferably different) , then two recombinational events may occur, and result in the exchange of a region of DNA between two DNA molecules.
  • Recombination may be "reciprocal,” and thus results in an exchange of DNA regions between two recombining DNA molecules.
  • it may be "non- reciprocal,” (also referred to as "gene conversion") and result in both recombining nucleic acid molecules having the same nucleotide sequence.
  • the frequency of recombination between two DNA molecules may be enhanced by treating the introduced DNA with agents which stimulate recombination. Examples of such agents include trimethylpsoralen, UV light, etc.
  • Gene targeting has also been used to correct an hprt deficiency in an hprt " ES cell line.
  • Cells corrected of the deficiency were used to produce chimeric animals. Significantly, all of the corrected cells exhibited gross disruption of the regions flanking the hprt locus; all of the cells tested were found to contain at least one copy of the vector used to correct the deficiency, integrated at the hprt locus (Thompson, S. et al.. Cell 56 . :313-321 (1989); Roller, B.H. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 8(5:8927-8931 (1989)).
  • the gene of interest In order to utilize the "gene targeting" method, the gene of interest must have been previously cloned, and the intron-exon boundaries determined. The method results in the insertion of a marker gene (i.e. the nptll gene) into a translated region of a particular gene of interest. Thus, use of the gene targeting method results in the gross destruction of the gene of interest.
  • chimeric mice carrying the homeobox hox 1.1 allele have been produced using a modification of the gene targeting method (Zimmer, A. et al.. Nature 338:150-154 (1989) . In this modification, the integration of vector sequences was avoided by microinjecting ES cells with linear DNA containing only a portion of the hox 1.1 allele, without any accompanying vector sequences.
  • FIG. 1A The use of the gene targeting method is illustrated in Figure 1A.
  • a gene construct is produced in which the nptll gene is inserted into an exon (designated region "3") of a sequence of the hprt gene.
  • the construct is then permitted to undergo recombination with the hprt gene of a cell.
  • Such recombination results in the replacement of the exon 3 sequence of the cell with the disrupted exon 3 - notII sequence of the construct.
  • the use of gene targeting to alter a gene of a cell results in the formation of a gross alteration in the sequence of that gene.
  • the efficiency of gene targeting is approximately 1/300.
  • the present invention is capable of producing subtle, precise, and predetermined mutations in the sequence of a desired gene of a cell.
  • the present invention has several embodiments, the simplest of which is illustrated in Figure IB.
  • an insertion vector is used to mutate the nucleotide sequence of the hprt gene.
  • the use of this vector type in combination with a second selectable reversion event prevents the disruption of the chromosome by the nptll gene or by the vector sequences.
  • gross distortions of the recipient chromosome are avoided by the present invention.
  • the efficiency of the gene targeting was substantially improved (i.e. 1/32 as opposed to 1/300) .
  • the DNA molecule(s) which are to be introduced into the recipient cell preferably contains a region of homology with a region of the cellular genome.
  • the DNA molecule will contain two regions of homology with the genome (both chromosomal and episomal) of the pluripotent cell. These regions of homology will preferably flank a "desired gene sequence" whose incorporation into the cellular genome is desired.
  • the regions of homology may be of any size greater than two bases long. Most preferably, the regions of homology will be greater than 10 bases long.
  • the DNA molecule(s) may be single stranded, but are preferably double stranded.
  • the DNA molecule(s) may be introduced to the cell as one or more RNA molecules which may be converted to DNA by reverse transcriptase or by other means.
  • the DNA molecule will be double stranded linear molecule.
  • such a molecule is obtained by cleaving a closed covalent circular molecule to form a linear molecule.
  • a restriction endonuclease capable of cleaving the molecule at a single site to produce either a blunt end or staggered end linear molecule is employed.
  • the nucleotides on each side of this restriction site will comprise at least a portion of the preferred two regions of homology between the DNA molecule being introduced and the cellular genome.
  • the invention thus provides a method for introducing the "desired gene sequence" into the genome of an animal or plant at a specific chromosomal location.
  • the “desired gene sequence” may be of any length, and have any nucleotide sequence. It may comprise one or more gene sequences which encode complete proteins, fragments of such gene sequences, regulatory sequences, etc.
  • the desired gene sequence may differ only slightly from a native gene of the recipient cell (for example, it may contain single, or multiple base alterations, insertions or deletions relative to the native gene) . The use of such desired gene sequences will permit one to create subtle and precise changes in the genome of the recipient cell.
  • the present invention provides a means for manipulating and modulating gene expression and regulation.
  • the invention provides a mean for manipulating and modulating gene expression and protein structure through the replacement of a gene sequence with a "non-selectable" "desired gene sequence.”
  • a gene sequence is non-selectable if its presence or expression in a recipient cell provides no survival advantage to the cell under the culturing conditions employed. Thus, by definition, one cannot select for cells which have received a "non-selectable” gene sequence.
  • a "dominant" gene sequence is one which can under certain circumstances provide a survival advantage to a recipient cell.
  • the neomycin resistance conferred by the nptll gene is a survival advantage to a cell cultured in the presence of neomycin or G418.
  • the nptll gene is thus a dominant, rather than a non-selectable gene sequence.
  • the invention permits the replacement of a gene sequence which is present in the recipient cell with an "analog" sequence.
  • a sequence is said to be an analog of another sequence if the two sequences are substantially similar in sequence, but have minor changes in sequence corresponding to single base substitutions, deletions, or insertions with respect to one another, or if they possess "minor" multiple base alterations. Such alterations are intended to exclude insertions of dominant selectable marker genes.
  • the consequence of the second recombinational event is to replace the DNA sequence which is normally present between the flanking regions of homology in the cell's genome, with the desired DNA sequence, and to eliminate the instability of gene replacement.
  • the DNA molecule containing the desired gene sequence may be introduced into the pluripotent cell by any method which will permit the introduced molecule to undergo recombination at its regions of homology. Some methods, such as direct microinjection, or calcium phosphate transformation, may cause the introduced molecule to form concatemers upon integration. These concatemers may resolve themselves to form non-concatemeric integration structures. Since the presence of concatemers is not desired, methods which produce them are not preferred.
  • the DNA is introduced by electroporation (Toneguzzo, F.
  • the cells After permitting the introduction of the DNA molecule(s) , the cells are cultured under conventional conditions, as are known in the art. In order to facilitate the recovery of those cells which have received the DNA molecule containing the desired gene sequence, it is preferable to introduce the DNA containing the desired gene sequence in combination with a second gene sequence which would contain a detectable marker gene sequence.
  • any gene sequence whose presence in a cell permits one to recognize and clonally isolate the cell may be employed as a detectable marker gene sequence.
  • the presence of the detectable marker sequence in a recipient cell is recognized by hybridization, by detection of radiolabelled nucleotides, or by other assays of detection which do not require the expression of the detectable marker sequence.
  • sequences are detected using PCR (Mullis, K. et al.. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H.
  • PCR achieves the amplification of a specific nucleic acid sequence using two oligonucleotide primers complementary to regions of the sequence to be amplified. Extension products incorporating the primers then become templates for subsequent replication steps.
  • PCR provides a method for selectively increasing the concentration of a nucleic acid molecule having a particular sequence even when that molecule has not been previously purified and is present only in a single copy in a particular sample.
  • the method can be used to amplify either single or double stranded DNA.
  • the detectable marker gene sequence will be expressed in the recipient cell, and will result in a selectable phenotype. Examples of such preferred detectable gene sequences include the hprt gene (Littlefield, J.W.
  • a xanthine-guanine phosphoribosyltransferase (gpt) gene, or an adenosine phosphoribosyltransferase (aprt) gene (Sambrook et al.. In: Molecular Cloning A Laboratory Manual. 2nd. Ed., Cold Spring Harbor Laboratory Press, NY (1989), herein incorporated by reference)
  • a tk gene i.e. thymidine kinase gene
  • the tk gene of herpes simplex virus Gibpes simplex virus
  • genes include gene sequences which encode enzymes such as dihydrofolate reductase (DHFR) enzyme, adenosine deaminase (ADA) , asparagine synthetase (AS) , hygromycin B phosphotransferase, or a CAD enzyme (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase) (Sambrook et al.. In: Molecular Cloning A Laboratory Manual. 2nd. Ed., Cold Spring Harbor Laboratory Press, NY (1989), herein incorporated by reference) .
  • DHFR dihydrofolate reductase
  • ADA adenosine deaminase
  • AS asparagine synthetase
  • hygromycin B phosphotransferase hygromycin B phosphotransferase
  • CAD enzyme carbamyl phosphate synthetase, aspartate trans
  • TK thymidine kinase
  • HPRT hypoxanthine-phophoribosyltransferase
  • XGPRT xanthine-guanine phosphoribosyltransferase
  • APRT adenosine phosphoribosyltransferase
  • Cells expressing active thymidine kinase are able to grow in media containing HATG, but are unable to grow in media containing nucleoside analogues such as 5-azacytidine (Giphart-Gassler, M. et al.. Mutat. Res. 214:223-232 (1989)).
  • Cells containing an active HSV-tk gene are incapable of growing in the presence of gangcylovir or similar agents.
  • the detectable marker gene may be any gene which can complement for a recognizable cellular deficiency.
  • the gene for HPRT could be used as the detectable marker gene sequence when employing cells lacking HPRT activity.
  • this gene is an example of a gene whose expression product may be used to select mutant cells, or to "negatively select" for cells which express this gene product.
  • the nptll gene (Southern, P.J., et al.. J. Molec. Appl. Genet. .1:327-341 (1982); Smithies, O. et al.. Nature 317:230-234 (1985), which references are incorporated herein by reference) is the most preferred detectable marker gene sequence. Constructs which contain both an nptll gene and either a tk gene or an hprt gene are especially preferred.
  • the detectable marker gene sequence flanked by the regions of homology, is provided to the recipient cells on the same DNA molecule which contains the desired gene sequence.
  • this DNA molecule be a linear molecule.
  • the DNA molecule will, in addition to the desired gene sequence, the flanking regions of homology and the detectable marker gene sequence, contain an additional gene sequence which will permit the selection or recognition of cells which have undergone the second recombinational event. This additional gene sequence will be excised from the cell's genome as a direct consequence of the second recombinational event.
  • gene sequences which are suitable for this purpose include any gene sequence whose loss from a cell can be detected or selected for.
  • Examples of such "negative selection" gene sequences include the hprt gene, and the tk gene (especially the t gene of herpes simplex virus) .
  • the frequency of the second recombinational event is approximately 10" 5 .
  • the use of a "negative selection" gene sequence permits one to identify such recombinant cells at a frequency of approximately 100%.
  • the DNA molecule may have a region of heterology located at the proposed insertion site. Insertion of such a vector permits one to select for recombinants which have recombined at the insertion site (and not at other potential sites) .
  • the region of heterology which may be introduced at the insertion site of the DNA molecule may be either short (for example, 26 base pairs, Figure 2B) or of substantial size (for example, 2 kb, Figure 2A) .
  • the site of linearization may be 5', 3' , or within the region of heterology. When the site of linearization is within the region of heterology, the efficiency of gene targeting is 1/63.
  • the region of heterology may be located at a site internal to the region of homology where the desired recombination shall occur. Such a construct can be used when one desires to introduce a subtle mutation into a locus of the cellular gene at a site other than that of the site of desired recombination.
  • the detectable marker gene sequence flanked by the regions of homology, will be provided to the recipient cell on a different DNA molecule from that which contains the desired gene sequence. It is preferred that these molecules be linear molecules.
  • the detectable marker gene sequence and the desired gene sequence will most preferably be provided to the recipient cell by co- electroporation, or by other equivalent techniques. After selection of such recipients (preferably through the use of a detectable marker sequence which expresses the nptll gene and thus confers cellular resistance to the antibiotic G418) , the cells are grown up and screened to confirm the insertion event (preferably using PCR) . In the absence of any selection, only one cell in IO 7 would be expected to have the predicted recombinant structures.
  • a detectable marker sequence such as the nptll gene
  • recipient cells which contain and express a detectable marker sequence (such as the nptll gene)
  • a detectable marker sequence such as the nptll gene
  • such enrichment enables one to identify the desired recipient cell (in which the introduced DNA has integrated into the cell's genome) by screening only 800 -1,500 cells. Such screening is preferably done using PCR, or other equivalent methods. Using such negative selection techniques, one may manipulate the vector copy number.
  • the two introduced DNA molecules will generally not have integrated into the same site in the genome of the recipient cell.
  • the desired gene sequence will have integrated in a manner so as to replace the native cellular gene sequence between the flanking regions of homology.
  • the locus of integration of the detectable marker gene is unimportant for the purposes of the present invention, provided it is not genetically linked to the same locus as the desired gene sequence. If desired, however, it is possible to incorporate a gene sequence capable of negative selection along with the DNA containing the detectable marker sequence. Thus, one can ultimately select for cells which have lost the introduced selectable marker gene sequence DNA.
  • each embodiment requires the screening of a significant number of candidate cells in order to identify the desired recombinant cell. It is, however, possible to directly select for the desired recombinant cell by employing a variation of the above embodiments.
  • This embodiment of the invention is illustrated in Figure 13. In the methods illustrated in Figure 13, if the sequence located below the asterisk is a neo gene, then only the mutant revertants will be selected if 6-thioguanine and G418 selection is applied to select for the excision events.
  • the method for direct selection of the desired cells relies upon the phenotypic difference in targeted and non- targeted cells and the use of a single gene which can be used for both positive and negative selection.
  • three populations of cells will be created.
  • the first class of cells will be those which have failed to receive the desired DNA molecule. This class will comprise virtually all of the candidate cells isolated on completion of the experiment.
  • the second class of cells will be those cells in which the desired gene sequence has been incorporated at a random insertion site (i.e. a site other than in the gene desired to be mutated) . Approximately one cell in 10 3 -10 4 total cells will be in this class.
  • the third class of cells will be those cells in which the desired gene sequence has been incorporated by homologous recombination into a site in the desired gene. Approximately one cell in 10 5 -10 6 total cells will be in this class.
  • the cells of the first class non-transfected cells
  • the cells of the third class may be selected from the cells of the second class (random insertions) if a phenotypic difference exists between the cells of the two classes.
  • the method comprises incubating a "precursor cell" (i.e.
  • a culturing condition i.e. medium, temperature, etc.
  • a culturing condition is said to be “non-selective” if it is capable of promoting the growth (or sustaining the viability) of a precursor cell, a desired cell, and an intermediate cell type (i.e. a cell obtained during the progression of a precursor cell into a desired cell) .
  • a culturing condition is said to be “selective” if it is capable of promoting the growth (or sustaining the viability) of only certain cells (i.e.
  • TK thymidine kinase
  • HPRT hypoxanthine-phophoribosyltransferase
  • XGPRT xanthine-guaninephosphoribosyltransferase
  • APRT adenosine phosphoribosyltransferase
  • cells that do contain such active enzymes are able to grow in such medium, but are unable to grow in medium containing nucleoside analogs such as 5-bromodeoxyuridine, 6-thioguanine, 8- azapurine, etc.
  • Such incubation is conducted in the presence of a DNA molecule containing a desired non-selectable gene sequence.
  • the DNA molecule additionally contains two regions of homology which flank this desired gene sequence, and which are sufficient to permit the desired gene sequence to undergo homologous recombination with a predetermined gene sequence of the genome of the precursor cell.
  • the DNA molecule additionally contains a selectable gene sequence whose presence or expression in the cell can be selected for by culturing the cell under a first set of selective culture conditions, and whose presence or expression in the cell can be selected against by culturing the cell under a second set of selective culture conditions.
  • selectable gene sequences include gene sequences which encode an active thymidine kinase (TK) enzyme, a hypoxanthine-phophoribosyltransferase (HPRT) enzyme, a xanthine-guaninephosphoribosyltransferase (XGPRT) enzyme, or an adenosine phosphoribosyltransferase (APRT) enzyme.
  • Such gene sequences can be used for both positive and negative selection.
  • Additional gene sequences which can be used as selectable gene sequences include those which encode enzymes such as dihydrofolate reductase (DHFR) enzyme, adenosine deaminase (ADA) , asparagine synthetase (AS) , hygromycin B phosphotransferase, or a CAD enzyme (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase) .
  • enzymes such as dihydrofolate reductase (DHFR) enzyme, adenosine deaminase (ADA) , asparagine synthetase (AS) , hygromycin B phosphotransferase, or a CAD enzyme (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase) .
  • Such gene sequences can be used only for positive selection.
  • the incubation is performed under conditions sufficient to permit the DNA molecule to be introduced into the precursor cell.
  • Such introduced DNA molecules are able to then undergo homologous recombination with the predetermined gene sequence of the genome of the precursor cell to thereby produce the desired cell wherein the desired non-selectable gene sequence has been inserted into the predetermined gene sequence.
  • Such a desired cell can be recovered by culturing the cell under the first set of selective culture conditions, by then permitting the cell to undergo intrachromosomal recombination under non-selective culture conditions, and by then incubating the cell under the second set of selective culture conditions.
  • the precursor cell lacks an activehypoxanthine-phophoribosyltransferase (HPRT) enzyme, a xanthine-guaninephosphoribosyltransferase (XGPRT) enzyme, or an adenosine phosphoribosyltransferase (APRT) enzyme, and the selectable gene sequence expresses an active HPRT, XGPRT or APRT enzyme.
  • HPRT activehypoxanthine-phophoribosyltransferase
  • XGPRT xanthine-guaninephosphoribosyltransferase
  • APRT adenosine phosphoribosyltransferase
  • the selectable gene sequence expresses an active HPRT, XGPRT or APRT enzyme.
  • medium containing hypoxanthine, aminopterin and/or mycophenolic acid and preferably adenine, xanthine, and/or thymidine
  • the precursor cell lacks an active TK enzyme, and the selectable gene sequence expresses an active TK enzyme.
  • a nucleoside analog such as 5-bromodeoxyuridine, 6-thioguanine, 8-azapurine, etc.
  • the precursor cell lacks an active TK enzyme, and the selectable gene sequence expresses an active TK enzyme.
  • the first set of selectable culture conditions medium containing hypoxanthine, aminopterin, and thymidine is employed.
  • a thymidine analog such as FIAU (Borrelli, Proc. Nat'l. Acad. Sci. (U.S.A.) 85:7572 (1988), or gangcyclovir, etc.
  • a preferred negative selectable marker is the hprt gene (cells expressing an active HPRT enzyme are unable to grow in the presence of certain nucleoside analogues such as 6- thioguanine, etc.).
  • 6-thioguanine as a negative selection agent, a density of IO 4 cells / cm 2 is preferably used since the efficiency of 6-thioguanine selection is cell density dependent.
  • a typical experiment with IO 7 transfected cells would yield approximately 10 revertant cells after successive selection.
  • the relative yield of revertant clones can be substantially increased by using "Poly A Selection" for the first round of selection. "Poly A Selection” is discussed in detail in Example 6 below.
  • the chimeric or transgenic animals of the present invention are prepared by introducing one or more DNA molecules into a precursor pluripotent cell, most preferably an ES cell, or equivalent (Robertson, E.J. , In: Current Communications in Molecular Biology. Capecchi, M.R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) , pp. 39-44, which reference is incorporated herein by reference) .
  • ES cell most preferably an ES cell, or equivalent
  • the term "precursor” is intended to denote only that the pluripotent cell is a precursor to the desired (“transfected") pluripotent cell which is prepared in accordance with the teachings of the present invention.
  • the pluripotent (precursor or transfected) cell may be cultured in vivo r in a manner known in the art (Evans, M.J. et al.. Nature 292:154-156 (1981)) to form a chimeric or transgenic animal.
  • Any ES cell may be used in accordance with the present invention. It is, however, preferred to use primary isolates of ES cells. Such isolates may be obtained directly from embryos such as the CCE cell line disclosed by Robertson, E.J. , In: Current Communications in Molecular Biology, Capecchi, M.R. (ed.). Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) , pp.
  • Clonally selected ES cells are approximately 10-fold more effective in producing transgenic animals than the progenitor cell line CCE.
  • clonal selection provides no advantage.
  • An example of ES cell lines which have been clonally derived from embryos are the ES cell lines, AB1 (hprt*) or AB2.1 (hprt°.
  • the ES cells are preferably cultured on stomal cells (such as STO cells (especially SNC4 STO cells) and/or primary embryonic fibroblast cells) as described by E.J. Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. (E.J.
  • the stomal (and/or fibroblast) cells serve to eliminate the clonal overgrowth of abnormal ES cells.
  • the cells are cultured in the presence of leukocyte inhibitory factor ("lif") (Gough, N.M. et al.. Reprod. Fertil. Dev. .1:281-288 (1989) ; Yamamori, Y. et al.. Science 246:1412-1416 (1989), both of which references are incorporated herein by reference) . Since the gene encoding lif has been cloned (Gough, N.M. et al.. Reprod.
  • ES cell lines may be derived or isolated from any species (for example, chicken, etc.), although cells derived or isolated from mammals such as rodents (i.e. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle, primates and humans are preferred.
  • the chimeric or transgenic plants of the invention are produced through the regeneration of a plant cell which has received a DNA molecule through the use of the methods disclosed herein. All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the introduced gene sequence.
  • Some suitable plants include, for example, species from the genera Fragaria. Lotus, Medicago, Onobrvchis. Trifolium. Trigonella. Vigna. Citrus. Linum. Geranium, Manicot, Daucus. Arabidopsis. Brassica. Raphanus. Sinapis. Atropa. Capsicum. Datura. Hyoscvamus. Lvcopersion. Nicotiana. Solanum.
  • Regeneration varies from species to species of plants, but generally a suspension of transformed protoplasts containing the introduced gene sequence is formed. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins.
  • glutamic acid and proline to the medium, especially for such species as corn and alfalfa.
  • Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
  • the mature plants, grown from the transformed plant cells are selfed to produce an inbred plant.
  • the inbred plant produces seed containing the introduced gene sequence. These seeds can be grown to produce plants that express this desired gene sequence.
  • Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention.
  • variant describes phenotypic changes that are stable and heritable, including heritable variation that is sexually transmitted to progeny of plants.
  • the DNA molecule(s) which are to be introduced into the recipient cells in accordance with the methods of the present invention will be incorporated into a plasmid or viral vector (or a derivative thereof) capable of autonomous replication in a host cell.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli such as, for example, pBR322, ColEl, pSClOl, pACYC 184, T ⁇ VX.
  • Such plasmids are, for example, disclosed by Maniatis, T. , et al. (In: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In:- The Molecular Biology of the Bacilli. Academic Press, NY (1982) , pp. 307-329) .
  • Suitable Streptomyces plasmids include pIJlOl (Kendall, K.J., et al.. J. Bacteriol. 169:4177-4183 (1987)), and Streptomyces bacteriophages such as ⁇ C31 (Chater, K.F., et al..
  • yeast vectors include the yeast 2- micron circle, the expression plasmids YEP13, YCP and YRP, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al.. Miami Wntr. Symp.
  • vectors which may be used to replicate the DNA molecules in a mammalian host include animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus.
  • the methods of the present invention permit the introduction of a desired gene sequence into an animal or plant cell.
  • the methods of the present invention may be used to introduce DNA into germ line cells of animals in order to produce chimeric or transgenic animals which contain a desired gene sequence.
  • the animals which may be produced through application of the described method include chicken, non-human mammals (especially. rodents (i.e. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle and non-human primates) .
  • the desired gene sequence may be of any length, and have any nucleotide sequence.
  • the sequence of the desired gene sequence in order to create single, or multiple base alterations, insertions or deletions in any preselected gene of a cell. If such changes are within a translated region of a native gene sequence, then a new protein variant of a native protein can be obtained.
  • Such a procedure can, for example be used to produce animals which produce improved (i.e. more stable, more active, etc.) enzymes, binding proteins, receptors, receptor ligands, etc.
  • the methods of the present invention may be used to produce cells in which a natural gene has been replaced with a heterologous gene.
  • a gene is said to be heterologous to a transgenic cell if it is derivable from a species other than that of the transgenic cell.
  • this replacement may be accomplished in a single step ( Figure 3) .
  • a DNA molecule containing a desired gene sequence and a region of homology is introduced into a recipient cell.
  • a selectable marker gene is also introduced into the cell, and used to select for cells which have underwent recombination.
  • the method results in the replacement of the normal sequences adjacent to the region of homology with the heterologous sequences of the desired DNA sequence.
  • this replacement may be accomplished in a two steps ( Figure 4) .
  • a cell is provided with a DNA molecule containing a desired gene sequence and a region of homology.
  • the DNA molecule also contains a selectable marker gene used to select for cells which have undergone a recombinational event that has resulted in the insertion of the introduced DNA molecule into their chromosomes at the site of homology.
  • the structure of such an insertion site is depicted in Figure 4A.
  • the introduced DNA molecule will also contain a "negative selectable" marker gene which can be used to select for cells which undergo a second recombinational event that results in the loss of the inserted DNA.
  • a second DNA molecule is employed to complete the gene replacement. This second DNA molecule need not contain any selectable marker gene.
  • a second recombinational event occurs which exchanges the "second" DNA molecule for the integrated "first" DNA molecule (including the desired DNA sequence, the selectable marker sequence, and the "negative selectable” marker sequence contained on that molecule) .
  • This aspect of the invention is illustrated in Figure 4B.
  • subtle mutations may be introduced into a desired locus using a "cassette" construct containing both a positive selection marker (such as the nptll gene or the gpt gene) and a negative selection marker (such as the tk gene) .
  • a positive selection marker such as the nptll gene or the gpt gene
  • a negative selection marker such as the tk gene
  • the method is applicable to any gene sequence. It is especially useful in producing cells which contain heterologous immunoglobulins (such as the heavy chain locus of an immunoglobulin) .
  • the first step in replacing a large region of a chromosome with a desired sequence involves setting up an initial target.
  • a recipient cell is provided with a DNA molecule which contains a "first fragment" of the total desired replacement sequence ( Figure 6A) .
  • This "first fragment" of the desired replacement sequence contains a selectable marker sequence (most preferably the nptll gene) at its end.
  • the DNA molecule also contains a "dual selection" gene sequence which encodes a non-functional fragment of a gene sequence for which both a positive and a negative selection exists.
  • gpt gene when used in the context of an hprt " cell.
  • Cells which express a functional gpt gene can be selected for by their ability to grow in HAT medium; Cells which lack a functional gpt gene can be selected for by their ability to grow in the presence of 6-thioguanine.
  • Homologous recombination results in the insertion of the DNA molecule into the cell's genome at the region of homology ( Figure 6A) .
  • this step results in the creation of a cell whose genome contains the selectable marker gene, it is possible to select for the desired recombinational event.
  • a second DNA molecule is provided to the cell.
  • This second DNA molecule contains a "second fragment" of the desired replacement sequence as well as a sequence of the dual selection gene that, due to an internal deletion, is incapable of encoding a functional gene product.
  • Homologous recombination results in the insertion of the second DNA molecule into the cell's genome in a manner so as to create a functional dual selection gene ( Figure 6B) .
  • Recombination also results in the integration of a non-functional fragment of the dual selection gene.
  • this step results in the creation of a cell whose genome contains a functional dual selection gene, it is possible to select for the desired recombinational event.
  • a third DNA molecule is provided to the cell.
  • This third DNA molecule contains both the "first" and “second" fragments of the desired replacement sequence. Homologous recombination results in the insertion of the third DNA molecule into the cell's genome in a manner so as to delete the functional dual selection gene.
  • the non-functional fragment of the dual selection gene (formed in step 2) is not affected by the recombination, and is retained ( Figure 6C) .
  • this step results in the creation of a cell whose genome lacks the dual selection gene, it is possible to select for the desired recombinational event.
  • a fourth DNA molecule is provided to the cell.
  • This fourth DNA molecule contains a "third fragment" of the desired replacement sequence as well as a sequence of the dual selection gene that, as in step 2, is incapable of encoding a functional gene product due to an internal deletion.
  • Homologous recombination results in the insertion of the fourth DNA molecule into the cell's genome in a manner so as to create a functional dual selection gene ( Figure 6D) .
  • Recombination also results in the integration of a non-functional fragment of the dual selection gene.
  • this step results in the creation of a cell whose genome contains a functional dual selection gene, it is possible to select for the desired recombinational event.
  • a fifth DNA molecule is provided to the cell.
  • This fifth DNA molecule contains both the "second" and “third” fragments of the desired replacement sequence. Homologous recombination results in the insertion of the fifth DNA molecule into the cell's genome in a manner so as to delete the functional dual selection gene.
  • the non-functional fragment of the dual selection gene (formed in step 4) is not affected by the recombination, and is retained ( Figure 6C) . Importantly, since this step results in the creation of a cell whose genome lacks the dual selection gene, it is possible to select for the desired recombinational event.
  • the net effect of the above- described steps is to produce a cell whose genome has been engineered to contain a "first,” “second,” and “third” "fragment” of a particular desired gene in a contiguous manner.
  • the steps may be repeated as desired in order to introduce additional "fragments" into the cell's genome.
  • cells can be constructed which contain heterologous genes, chromosome fragments, or chromosomes, that could not be introduced using a single vector.
  • each step of the method can be selected for.
  • this aspect of the present invention may be used to produce "humanized" antibodies (i.e. non-human antibodies which are non-immunogenic in a human) (Robinson, R.R. et al..
  • the method may also be used to produce animals having superior resistance to disease, animals which constitute or produce improved food sources, animals which provide fibers, hides, etc. having more desirable characteristics.
  • the method may also be used to produce new animal models for human genetic diseases.
  • the method may be used to "humanize" the CD4 analog of an animal, and thus provide an animal model for AIDS.
  • animal models can be used for drug testing, and thus hasten the development of new therapies for genetic diseases.
  • the present invention permits the formation of cells and of transgenic animals which contain mutations in medically or clinically significant heterologous genes.
  • a gene is said to be medically or clinically significant if it expresses an isotype of a protein associated with a human or animal disease or condition.
  • genes include the genes which encode: topoisomerase pl80, 5- ⁇ : reductase, ACAT, 5-lipoxygenase, the insulin receptor, the interleukin-2 receptor, the epidermal growth factor receptor, the seratonin receptor, the dopamine receptor, the GABA receptor, the V 2 vasopressin receptors, G proteins (signal transduction) , phospholipase C proteins, and insulin.
  • a transgenic mouse produced by microinjection which expresses human insulin was reported by Selden, R.F. et al. (European Patent Publication No. 247,494, which reference is incorporated herein by reference) .
  • the transgenic cells and animals discussed above can be used to study human gene regulation.
  • transgenic animals which express a human isotype of topoisomerase pl80, 5- ⁇ reductase, ACAT,5-lipoxygenase, or hormone or cytokine receptors would have ultility in in vivo drug screening.
  • the expression of topoisomerase pl80 is associated with resistance to chemotherapeutics.
  • agents which interfere with this enzyme could be used to enhance the effectiveness of chemotherapy.
  • An animal, especially a rat, capable of expressing a human isotype of 5- ⁇ reductase (especially in the prostate gland) would be highly desirable.
  • ACAT is a key enzyme in lipid metabolism; an animal model for its regulation would be extremely valuable.
  • mice that express 5-lipoxygenase could be of interest to many research programs, particularly to screen isotype selective inhibitors.
  • An animal which expressed human hormone or cytokine receptor proteins would be valuable in identifying agonists and antagonists of receptor action.
  • an animal that expressed components of the human signal transduction system i.e. G proteins and phospholipase Cs, etc.
  • G proteins and phospholipase Cs, etc. could be used to study the pathophysiologic consequences of disordered function of these proteins.
  • the present invention can be used to produce cells and animals which express human isotypes of transport proteins (i.e. proteins which facilitate or enable the transport of other molecules or ions across membranes in the gut, blood brain barrier, kidney, etc.). Such cells or animals can then be used to study the role of such proteins in metabolism.
  • the extent and patterns of conjugation mediated by such isotypes may be studied in order to investigate the pharmacokinetic consequences of specific differences in protein structure or sequence.
  • Glucoronide transferase, glycine conjugation and sulfation, methylases, and glutathione conjugation are examples of enzymes of particular interest in this regard.
  • the clearance of many compounds is mediated by esterases. Cells or animals which express heterologous isotypes of such esterases may be exploited in investigating such clearance. Cells or animals which express isotypes of proteins involved in azo or nitro reduction would be desirable for research on the processes of azo or nitro reduction.
  • potential therapeutic agents are frequently found to induce toxic effects in one animal model but not in another animal model.
  • the present invention permits one to produce transgenic cells or animals which could facilitate such determinations.
  • the methods of the present invention may be used to produce alterations in a regulatory region for a native gene sequence.
  • the invention provides a means for altering the nature or control of transcription or translation of any native gene sequence which is regulated by the regulatory region. For example, it is possible to introduce mutations which remove feedback inhibition, and thus result in increased gene expression. Similarly, it is possible to impair the transcriptional capacity of a sequence in order to decrease gene expression.
  • Such alterations are especially valuable in gene therapy protocols, and in the development of improved animal models of human disease.
  • the capacity to increase insulin gene transcription or translation provides a potential genetic therapy for diabetes.
  • the ability to impair the synthesis of beta globin chains provides an animal model for beta- thalassemia.
  • the methods of the present invention may be used to investigate gene regulation, expression and organization in animals. Since the methods of the present invention utilize processes of DNA repair and recombination, agents which inhibit or impair the present methods may act by affecting these processes. Since agents which impair DNA repair and recombination have potential antineoplastic utility, the present invention provides a means for identifying novel antineoplastic agents.
  • the present invention may additionally be used to facilitate both the cloning of gene sequences, and the mapping of chromosomes or chromosomal abnormalities. Since the desired gene sequence need not be homologous or analogous to any native gene sequence of the recipient cell, the methods of the present invention permit one to produce animals which contain and express foreign gene sequences. If the cell expresses an analogous gene, the desired gene sequence may be expressed in addition to such analogous cellular genes (for example, an animal may express both a "humanized" receptor and an analogous native receptor) .
  • the invention provides a means for producing animals which express important human proteins (such as human interferons, tissue plasminogen activator, hormones (such as insulin and growth hormone) , blood factors (such as Factor VIII), etc.).
  • the methods of the invention may be used to introduce DNA into plant cells which can then be manipulated in order to produce chimeric or transgenic plants.
  • the plants which may be produced through application of the disclosed method include all multicellular, higher (i.e. non-fungal) plants.
  • a non- fungal plant is any plant which is not a fungus or yeast.
  • the methods of the invention may be used to introduce DNA into the somatic cells of an animal (particularly mammals including humans) or plant in order to provide a treatment for genetic disease (i.e. "gene therapy") .
  • genetic disease i.e. "gene therapy”
  • the principles of gene therapy are disclosed by Oldham, R.K. (In: Principles of Biotherapy. Raven Press, NY, 1987) , and similar texts.
  • the genetic lesion which causes the disease is replaced with a gene sequence encoding a preferred gene product.
  • Such genetic lesions are those responsible for diseases such as cystic fibrosis, phenylketonuria, hemophilia, von Willebrand's Disease, sickle cell anemia, thalassemia, galactosemia, fructose intolerance, diseases of glycogen storage, hyper- cholesterolemia, juvenile diabetes, hypothyroidism, Alzheimer's Disease, Huntington's Disease, Gout, Lesch-Nyhan Syndrome, etc. (Bondy, P.K. et al.. In: Disorders of Carbohydrate Metabo1ism. pp 221-340, Saunders (1974) ; Coleman, J. et al.. Molecular Mechanisms of Disease. Yale University Press, (1975)).
  • diseases such as cystic fibrosis, phenylketonuria, hemophilia, von Willebrand's Disease, sickle cell anemia, thalassemia, galactosemia, fructose intolerance, diseases of glycogen storage, hyper- cholesterolemia, juvenile diabetes,
  • the methods of the invention may be used to provide a treatment to protect recipient animals or plants from exposure to viruses, insects or herbicides (in the case of plants) , insecticides, toxins, etc.
  • the introduced gene would provide the recipient with gene sequences capable of mediating either an enhanced or novel expression of an enzyme, or other protein, capable of, for example, degrading an herbicide or toxin.
  • a plant cell may receive a gene sequence capable of mediating an enhanced or novel expression of a chitinase, thus conferring increased resistance to insect parasites.
  • pharmaceutically acceptable carriers i.e. liposomes, etc.
  • Such gene sequences can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • compositions suitable for effective administration will contain an effective amount of the desired gene sequence together with a suitable amount of carrier vehicle. Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the desired gene sequence (either with or without any associated carrier) .
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • appropriate macromolecules for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate
  • concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate the agent into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellu- lose or gelatine-micro ⁇ apsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • the methods of the present invention may be used to improve the food or fiber characteristics of plants or non-human animals.
  • the methods can be used to increase the overall levels of protein synthesis thereby resulting in faster growing plants or non-human animals, or in the production of plants and non-human animals which have increased food value.
  • Electroporation was performed as follows:
  • DNA used for electroporation was purified by CsCl gradient centrifugation.
  • a large-scale digest of this purified DNA was prepared by incubating the DNA with an appropriate restriction enzyme.
  • the large-scale digest was examined for complete digestion by running 500 ng on a minigel.
  • the DNA concentration of the large-scale digest should be no higher than 1 ⁇ g/ ⁇ l.
  • the large-scale digest was then extracted once with an equal volume of phenol/chloroform and once with an equal volume of chloroform.
  • the DNA was precipitated with 2.4 volumes of ethanol, pelleted by centrifugation, and dried using a Speed-Vac.
  • the pelleted DNA was then resuspended at the desired concentration (usually 1 ⁇ g/ ⁇ l) in a sterile Tris-EDTA buffer such as 0.1X TE (25 ⁇ l of DNA per electroporation). The concentration of the DNA was then measured with a fluorometer.
  • Embryonic stem cells of the AB1 cell line were cultured to approximately 80% confluence according to the methods of E.J. Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. (E.J. Robertson, Ed.), IRL Press, Oxford, 1987, pp 71-112). Cells were cultured in the presence of stomal cells which expressed lif into the culture medium. Cells were passaged 1:2 the day before electroporation, and fed 4 hours before harvesting. Cells were harvested by trypsinizing the cells, and by resuspending in media (cells from 2 x 10 cm plates were combined in a total volume of 10 ml in a 15 ml tube) .
  • the cells were pelleted by centrifugation, and the supernatant was removed by aspiration.
  • the cells were then resuspended in 10 ml of phosphate buffered saline and the total number of cells was determined by counting a 20 ⁇ l aliquot. The usual yield is 30 x 10 6 cells per 10 cm plate.
  • the cells were then pelleted by centrifugation and the supernatant was removed by aspiration. Cells were resuspended at a density of 11 x 10 6 cells/ml. A 20 ⁇ l aliquot was counted to confirm this cell density.
  • the time constant should read between 5.6 and 7.0.
  • the cuvette was left at room temperature for 5 minutes and then the cells were plated at an appropriate density (up to 2 x IO 7 cells/100 mm plate or 6 x 10 6 cells/60 mm plate) .
  • this cell density should not be exceeded since G418 takes 3-4 days before killing starts and plates will become over-confluent.
  • G418 selection was to be applied, it is applied 24 hours post-electroporation. G418 concentration must be titrated for every batch. The plate(s) were re-fed with fresh media + G418 every day for the first 6-7 days (until colonies are visible and most cell debris has been removed).
  • ES cells embryonic stem (“ES") cells were co-electroporated with a 4.5 kb nptll-containing vector (pPGKneobpA) which had been linearized by treatment with Xhol restriction endonuclease, and with the 6.5 kb HPRT vector, AD 8 (linearized with Sad) ( Figure 7) .
  • Electroporation 230 V, 500 ⁇ F were done on 0.9 ml aliquot of CCEp24 cells (7.5 x 10 6 cells/ml). The electroporation reactions were conducted using molar ratios of 1:1, 1:10, and 1:100 (notII DNA:HPRT DNA). The total amount of DNA provided was either 25, 50, 100, or 200 ⁇ g.
  • the vectors used in this experiment are illustrated in Figure 7. The results of this experiment are shown in Table 1.
  • Reversion of the hprt clones was done by measuring HAT R .
  • Cells were clonally expanded under 6-TG selection to prevent "jackpot" effects caused by the early recombinational loss of the duplicated element giving rise to a large number of colonies by cell division.
  • 10 7 cells were obtained, the cells were reseeded onto 90 mm plates without selection for 48 hours. After 48 hours HAT selection was applied and resistant colonies were scored 10 days later, typically 20 to 200 colonies were observed per IO 7 cells plated (Table 4) . Every clone examined reverted at a similar frequency.
  • RV Replacement Vector
  • IV Insertion Vector
  • the vectors contain exons 7, 8, and 9 of the hprt gene.
  • the polyadenylation site is located in exon 9.
  • a HinDIII site is present within exon 9, and an EcoRI site is located after the end of the exon.
  • the first vector employed contained a 5.0 kb region, and thus contained the polyadenylation site of exon 9 (Vector 6, Figure 10).
  • the frequency of insertion was high (i.e.
  • the methods of the present invention were further illustrated by their use to produce cells having precise and subtle mutations in the c-src locus of ES cells.
  • the c-src locus contains several exons, which are designated as "boxed" regions 2 and 3' in Figure 11.
  • the natural allele of exon 3' does not contain a Hindlll site.
  • the sequence of a portion of exon 3' is shown in Figure 11C.
  • Figure 11C As shown in Figure 11C, a 9 bp insertion into this exon will result in the formation of a HinDIII site.
  • a vector src 14 was prepared.
  • the src 14 vector is homologous to a region of the c-src locus.
  • the exon 3' sequence of the vector has been altered to contain the 9 base pair insertion needed to create a Hindlll site ( Figure 11C) .
  • the src 14 vector was introduced into ES cells by co- electroporation with a second vector (PGKneo) that contained the nptll gene, at a total DNA concentration of 25 ⁇ g/ml and a molar ratio of 1:5 (neo vector to targeting vector) in the manner described above.
  • exon 3" of the c-src gene of an ES cell was mutated to contain two different substitution mutations.
  • the natural allele of exon 3" does not contain either an Nhel site or an EcoRI site.
  • the replacement of the natural sequence ACC TGG TTC of exon 3" with the sequence TAG CTA GCT will result in the formation of an Nhel site.
  • replacement of ACA with GAA in exon 3" will create an EcoRI site ( Figure 12C) .
  • a vector (src 33) was prepared.
  • the src 33 vector is homologous to a region of the c-src locus.
  • the exon 3" sequence of the vector has been altered to contain the substitutions indicated above ( Figure 12C) .
  • the src 33 vector was introduced into ES cells by electroporation, in concert with a second vector that contained the nptll gene, in the manner described above. Cells were cultured in the presence of G418 in order to select for recombinant cells in which the nptll gene had integrated.

Abstract

Procédé de production de cellules animales contenant une séquence de gène voulue ayant été insérée dans une séquence de gène prédéterminée par recombinaison homologue. Le procédé permet la production de cellules animales présentant des modifications subtiles et précises de séquence et d'expression de gènes.
PCT/US1991/004006 1990-06-12 1991-06-07 Procede de recombinaison homologue dans des cellules animales et vegetales WO1991019796A1 (fr)

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JPH05507853A (ja) 1993-11-11
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EP0535144A4 (en) 1993-08-11
CA2084774A1 (fr) 1991-12-13
AU654284B2 (en) 1994-11-03

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