WO1993023553A1 - Production of transgenics by joining regulatory and coding regions in vivo - Google Patents

Abstract

A method for introducing into a cell an operably unlinked regulatory region and coding region that have been constructed such that they are able to join in vivo to form an operably linked unit is described. Prior to introduction into the cell, the ends of the regulatory region and the coding region are constructed to contain, for example, blunt ends such that blunt end ligation can occur, single stranded cohesive ends that are complementary to each other such that annealing between the strands can occur, or homologous sequence regions such that homologous recombination is able to occur. Transgenic organisms, cells and cell lines are also provided.

Description

PRODUCTION OF TRANSGENICS BY JOINING REGULATORY AND CODING REGIONS IN VIVO

Field of the Invention This invention relates to a versatile method for joining different regulatory regions with different coding regions in vivo, and to transgenic organisms, cell lines and cells relating thereto.

Background of the Invention A variety of methods have been employed to genetically alter eukaryotic genomes. DNA molecules have been introduced into cultured cells by calcium phosphate precipitation or electroporation, wherein the DNA enters the cell cytoplasm, with a fraction of the molecules entering the nucleus. Graham and Van der Ebb (1973) Virology 52: 456-467; Perucho et al. (1980) Cell 22: 9-17; Chu et al. (1987) Nucl. Acids Res. 15: 1311-1326; Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987); and Bishop and Smith (1989) Mol . Biol ■ Med. 6 : 283-298. DNA molecules have also been introduced into the nucleus of cells in culture or within a zygote by direct microinjection. Gordon et al . (1980) Proc. Nat. Acad. Sci . USA 77: 7380-7384; Gordon and Ruddle (1983) Methods in Enzymology 101: 411-433; and Wagner and Hoppe (1989) US Patent #4,873,191. And, DNA molecules have been incorporated into animal genomes via retroviral vector infection. Jaenisch et al. (1981) Cell 24: 519; Soriano et al. (1986) Science 234: 1409-1413; Stewart et al . (1987) EMBO J. 6 383-388. All of the above methods have a common disadvantage in that the promoter region of a gene that is intended to be introduced into the genome is directly linked to the coding region for a gene that is being introduced. This linkage requires additional cloning steps that increase the complexity and time required for such vector constructions. Moreover, it is frequently the case that one does not know prior to intreduction of a gene into a cell which combination of promoter with coding sequence will perform in the desired manner. This uncertainty is a particularly acute problem in the construction of transgenic eukaryotes where promoters can fall under the influence of unknown control elements upon integration into the eukaryotic genome. Allen et al. (1988) Nature 333: 852-855; Gossler et al. (1989) Science 244: 463-465; and Reddy et al. (1991) J. Virol. 65_.: 1507-1515.

Summary of the Invention

According to the invention, a method of treating cells to add a desired characteristic which may be predetermined is provided. A regulatory region and a coding region that are free of operable linkage are introduced into a cell. These regions are constructed and arranged such that they can join in vivo to form an operably linked unit. The regulatory region and coding region are permitted to join in vivo to form such an operably linked unit, which aids in obtaining said desired characteristic.

Variations of the method are introducing multiple regulatory regions and/or multiple coding regions into the cell. The method of the invention preferably has on the downstream end of the regulatory region and the upstream end of the coding region blunt DNA ends, single stranded cohesive DNA ends that are complementary to each other, or homologous sequence regions.

The method is preferably used to produce a transgenic non-human organism containing the operably linked regulatory region and coding region. The method may also be used for changing the genotype of a non-human organism to produce an abnormal condition in that organism which simulates a condition sometimes occurring in nature, or the method may be used for changing the genotype of a non-human organism derived at least partially from a treated cell so as to obtain a desired characteristic in the organism, for example, enhanced milk production, disease resistance, growth enhancement, enhanced nutritional value or production of a desired protein, preferably in desired tissues at desired times.

In addition, transgenic non-human organisms which have added a desired characteristic by the method of this invention, are provided. Cells and cell lines derived from such transgenic organisms, as well as cells and cell lines derived from the initial recipient cell of this invention, which have added a desired characteristic, are also provided.

It is an object of the invention to modify the phenotype and/or genotype of cells or transgenic organisms.

It is another object of the invention to minimize the number of cloning steps, and thereby reduce the complexity and time required for construction of vectors containing different: regulatory regions and coding regions.

It is yet another object of the invention to provide an uncomplicated method whereby a particular coding region can be put under the control of a particular regulatory region such that the coding region is expressed in a manner so as to produce a desired product, in the desired amount, in the desired tissues, and at the desired time.

It is yet another object of the invention to provide a method whereby panels of different regulatory regions and coding regions can be easily screened for recombinant molecules made up of a specific regulatory region joined to a specific coding region to produce a product that is expressed in a desired manner.

These and other aspects of the invention, as well as advantages of the invention, will be more apparent from the following detailed description of the invention. Brief Description of the Drawings

Figure la depicts the ligation of blunt ends on the downstream end of a CMV promoter region and the upstream end of a β-galactosidase coding region where the promoter region and the β-galactosidase coding region are on different DNA segments. Figure lb depicts the ligation of blunt ends on the downstream end of a CMV promoter region and the upstream end of a β-galactosidase coding region where the promoter region and the β-galactosidase coding region are on the same DNA segment-

Figure 2 depicts the addition of a poly(dC) tail to a CMV promoter region, and the addition of a poly(dG) tail to a β-galactosidase coding region in the presence of terminal transferase, and the subsequent annealing of these complementary tails.

Figure 3 depicts the generation of single stranded cohesive ends that are complementary to each other as a result of exonuclease III digestion of homologous sequence regions on the downstream end of a CMV promoter region and the upstream end of a β-galactosidase coding region, and the subsequent annealing and repair of these complementary sequences.

Figure 4 depicts homologous recombination between a CMV promoter region which contains a sequence region at its downstream end and a β-galactosidase coding region which contains a sequence region at its upstream end which is homologous to the promoter sequence region.

Figure 5 depicts the plasmid pCMVβ with some of its restriction endonuclease sites noted.

Figure 6 depicts a recombinant between a regulatory region and a coding region, and primers that are to be used in PCR amplification analysis that are upstream and downstream from the junction formed as a result of the joining of the regulatory and coding regions.

Figure 7 depicts a 4.2 kb fragment which includes the regulatory region for the rat α-tropomyosin gene.

Figure 8 depicts the coding regions for the myc and ras oneogenes. Detailed Description

This invention provides a versatile method for hooking up different regulatory regions with different coding regions in vivo, and thereby enabling modification of the genomes of non-human transgenic organisms or chimeric organisms, or of cells or cell lines, such that a desired characteristic, which may be predetermined, is added.

The term organism is meant to include animals, plants and fungi, or an organized group of cells from animals, plants or fungi, such as a tissue or an organ or parts thereof. Animals include fish, amphibians, birds and mammals. Preferred organisms are mammals and preferred mammals are rodents, rabbits, horses, cows, goats, sheep, cats, dogs, monkeys and humans. The term transgenic organism is meant to include an organism that is genetically altered by the introduction of DNA or RNA into its cells. By introduction of DNA it is meant that DNA or RNA molecules are caused to be added to the cell of the non-transgenic target organism. The term chimeric organism is an organism in which some of its cells have gained genetic information from the introduction of DNA into cells of the organism, or an ancestor of the organism, preferably at an embryonic stage.

Conventional recombinant DNA cloning methods require that the joining of the desired regulatory region with the desired coding region be constructed in vitro. Such in vitro vector constructions are time consuming and repetitive. This invention reduces the complexity and number of cloning and purification steps that would normally be required for such in vitro constructions. In one embodiment of the invention, panels of different regulatory regions and panels of different coding regions that are operably unlinked to each other are constructed, but are not further processed _in vitro to produce operably linked units. Rather, some combination of these panels is introduced into a cell where they join in vivo to form various operably linked units. An operably linked unit becomes incorporated into the genome of the cell or is maintained in the cell as an autonomously replicating unit.

A regulatory region is a region through which transcription of a gene is controlled. Such regulatory regions include a cis-acting DNA sequence. A function of this sequence is to be recognized by regulatory elements such as proteins. A regulatory region includes promoters and enhancers. A promoter is a DNA sequence which signals the start of RNA synthesis. An enhancer is a DNA sequence which alters the efficiency of transcription. It can direct the cell or cell type in which the adjacent promoter can function. The regulatory region may be derived from a regulatory region that is normally endogenous to the target cell, or from a regulatory region that is exogenous to the target cell. By endogenous regulatory region it is meant a regulatory region that is normally found in the genome of the target cell. By exogenous regulatory region, it is meant a regulatory region that is not normally found in the genome of the target cell.

The term coding region is a sequence of DNA that encodes for a product and that is free of a regulatory region. The coding region can code for any RNA or polypeptide product. The product may be a full length gene product or it may be a subfragment thereof, or it may be part of a fusion product. In the case of interrupted eukaryotic genes, a coding region can be sequences which include exonε and introns, as well as those which include exons and some introns, or only exons. The coding region may be derived from a coding region that is normally endogenous to the target cell, or from a coding region that is exogenous to the target cell.

The invention thus embraces in vivo joining of: a promoter to a coding region; and an enhancer already joined to a promoter to a coding region. Preferably the operably linked unit formed by this single joining event results in an expression system coding a full length gene product. Various combinations of regulatory regions and coding regions can be introduced into the cell. For example, one regulatory region may be joined with one coding region. According to an important aspect of this invention, other combinations may be employed: (i) multiple regulatory regions (at least two of the regulatory regions being different) with one coding region; (ii) one regulatory region with multiple coding regions (at least two of the coding regions being different); or (iii) multiple regulatory regions (at least two of the regulatory regions being different) with multiple coding regions (at least two of the coding regions being different). Preferably, the regulatory region and the coding region comprise separate molecules prior to their introduction into a cell. Alternatively, both the regulatory region and the coding region are part of the same DNA molecule, but are not operably linked because of a double stranded DNA cut between the regulatory region and coding region. In either embodiment, as a result of the in vivo joining event, a regulatory region becomes operably linked with a coding region.

The term operably linked unit means the situation where expression of the coding region is under the control of the regulatory region. Such control includes control by at least the promoter of the regulatory region. The regulatory region and/or the coding region may be wild type or may contain some type of mutation.

The term join means any process whereby the regulatory region and the coding region are physically connected so as to form an operably linked unit. Such a process includes joining of double stranded blunt DNA ends, joining of single stranded cohesive DNA ends, recombination between homologous regions of DNA, and non-homologous recombination. In vivo joining is able to occur under ordinary conditions of cell growth. Any temperature that is not destructive to the cell can be used. The ά-n vivo joining is believed to occur substantially instantaneously. In one embodiment of the invention, the regulatory region and coding region are constructed .in vitro such that the downstream end of the regulatory region and the upstream end of the coding region have blunt DNA ends. By downstream it is meant sequences proceeding farther in the direction of mRNA transcription. By upstream it is meant sequences that are farther in the direction opposite of mRNA transcription. For example, the coding region is downstream of the promoter, and the promoter is upstream of the coding region.

Upon introduction of the blunt end regions into the cell, in vivo ligation is able to occur. Blunt end ligation is a reaction that joins two double stranded DNA molecules directly at their ends. A DNA molecule that has blunt ends lacks any protruding single strands. For example, when DNA has been cleaved with a restriction enzyme that cuts across both DNA strands at the same position, blunt end ligation can be used to join the fragments directly together. Preferably, cleavage occurs downstream of the regulatory region and upstream of the initiator methionine codon of the coding region. See Figures la and lb where a blunt end that is generated on the downstream end of a CMV promoter region is ligated with a blunt end that has been generated on the upstream end of a β-galactosidase coding region to form an operably linked unit. The arrow depicted in the CMV promoter region indicates the direction of mRNA transcription. The advantage of this blunt end ligation technique is that any pair of ends may be joined together irrespective of sequence. Thus, two defined sequences can be joined together without introducing any additional material between them.

In another embodiment of the invention, the joining of the two DNA regions can be accomplished by generating complementary sequences on the ends of each of the regions, so that they can recombine into an operably linked unit when introduced together in vivo. The complementary sequences are of sufficient length and sequence content to promote association of the regulatory region with the coding region in vivo. The generation of complementary sequences can be accomplished in many ways. For example, a restriction enzyme may be used that makes staggered cuts to generate short, complementary single-stranded cohesive ends. This complementarity allows the cohesive ends to anneal by base pairing. Preferably, the single stranded DNA ends have a high G+C content because such ends favor a more stable association. The term high G+C content is meant to include a DNA sequence in which greater than 50% of its nucleotides are cytidylic acid and guanylic acid. Extensive lists of restriction endonucleases with the properties described here are described in catalogs from suppliers of molecular biology reagents: New England Biolabs, Beverly, MA 01915-5599; Stratagene, LaJolla, CA 92037; Promega, Madison, WI 53711-5399.

In another embodiment, the enzyme terminal transferase may be used. For example, the precursor dCTP is added in the presence of terminal transferase to generate a stretch of poly(dC) to the 3' ends of the regulatory region DNA. In the same way, dGTP is added to generate poly(dG) to the 3' ends of the coding region DNA. The poly(dC) tail anneals with the poly(dG) tail. Alternatively, poly(dG) can be added to the regulatory region DNA and poly(dC) can be added to the coding region DNA. The pair of deoxynucleotide triphosphates dATP and dTTP may also be used. The preferred deoxynucleotide triphosphates are dGTP and dCTP. See Figure 2 in which a poly(dC) tail that is added downstream of the CMV promoter in the presence of terminal transferase and a poly(dG) tail that is added upstream of the β-galactosidase coding region in the presence of terminal transferase, anneal to form an operably linked unit. This method has the advantage that the regulatory region associates only with the coding region and not with other regulatory region molecules, and the coding region associates only with the regulatory region and not with other coding region molecules. In a cell in which such a joining event has occurred, the cell is substantially free of regulatory regions joined to regulatory regions, and of coding regions joined to coding regions.

Complementary sequences may also be generated by exonuclease digestion of homologous sequence regions that are present on the downstream end of the regulatory region and the upstream end of the coding region. The term homologous sequence region is intended to include sequences of double stranded DNA that contain substantially the same nucleotide sequences. For example, complementary strands of the homologous sequence regions are revealed by digestion of each separate molecule with exonuclease III, a processive exonuclease that removes nucleotides from the 3' terminus of double stranded DNA. Preferably, by controlling the time of exonuclease digestion, a similar number of nucleotides can be removed from the regulatory region and coding region molecules, thereby revealing single stranded cohesive ends that associate with each other in an intracellular environment. See Figure 3 in which single stranded sticky ends are generated in a homologous sequence region, depicted by the stippled box between the Xhol and NotI restriction endonuclease sites on the downstream end of a CMV promoter region, and in a homologous sequence region between the Xhol and NotI sites on the upstream end of a β-galactosidase coding region, in the presence of exonuclease III. These sticky ends anneal to form an operably linked unit.

In yet another embodiment of the invention, the joining of the regulatory region and the coding region is accomplished by constructing in vitro a regulatory region with a sequence at its downstream end that is homologous to a sequence at the upstream end of the coding region, such that the homologous regions are able to undergo homologous recombination with each other in vivo. The sequence region should be of a length that is sufficient to permit i-n vivo homologous recombination to occur. See Figure 4 in which a homologous sequence region, depicted by the stippled box between the Xhol and NotI restriction endonuclease sites on -li¬

the downstream end of a CMV promoter region, and a homologous sequence region between the Xhol and NotI sites upstream of a β-galactosidase coding region, undergo homologous recombination to form an operably linked unit.

In still another embodiment of the invention, the joining of the two DNA regions is accomplished by in vivo processing of the ends that when introduced are non-homologous, non-blunt and non-complementary. Such joining is known as illegitimate recombination. This joining is possible if sufficient copies of the two DNA regions are introduced into a cell, although the potential for undesired regulatory region-regulatory region joining and coding region-coding region joining is enhanced.

The physical form of the DNA that is introduced into the cell includes supercoils, relaxed circles or linear DNA molecules. These DNA molecules can be intact or can contain one or more nicks. The preferred form is linear DNA. The regulatory and coding region DNA molecules may contain additional DNA sequences. For example, the regulatory or coding region may be part of a vector. Vectors include viruses, plasmids, cosmids, and YACS.

The DNA molecules can be introduced into the cell by any process which results in DNA uptake including microinjection, electroporation, or retroviral infection. Graham and Van der Ebb (1973) Virology 52: 456-467; Perucho et al. (1980) Cell 22: 9-17; Chu et al. (1987) Nucl■ Acids Res. 15: 1311-1326; Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987); and Bishop and Smith (1989) Mol Biol. Med. 6: 283-298. Introduction of the DNA into a cell includes introduction into cultured cells, tissues or the whole or part of a living organism. It also includes introduction into a single cell derived from a somatic or germ cell, into a zygote, into an embryonic stem cell, into a two-cell embryo, into a cell from another early stage embryo, or into a cell from a late stage embryo. Preferably, the DNA is introduced into a stem cell. Stem cells include totipotent cells and pluripotent cells. A totipotent cell means a cell that can differentiate into any cell or tissue type (e.g. an embryonic stem cell or a fertilized egg) . A pluripotent cell means a cell that can differentiate into many, but not all, cell or tissue types. An example of a pluripotent cell is a hematopoietic stem cell. In the embodiment where a transgenic non-human organism is allowed to develop, it is preferable that the DNA is injected into the pronucleus of a zygote or is introduced into embryonic stem cells.

In the embodiment of the invention where the regulatory region is on a separate molecule from the coding region, the two molecules can be introduced at the same time or separately. If they are introduced separately, they must be introduced within a close enough time span such that in vivo joining of the two molecules is able to occur. Preferably, the two molecules are introduced into the cell at the same time.

The joined regulatory region and coding region: can be incorporated into the genome of the cell; can be part of an autonomously replicating unit in the cell; or can persist in the cell in a form that is neither incorporated into the genome nor part of an autonomously replicating unit and can be partitioned into progency cells without duplication ("persistent DNA"). An example of an autonomously replicating unit is bovine papilloma virus. Elbrecht et al. (1987) Mol. Cell. Biol. 7, 1276-1279. Preferably, the operably linked unit is incorporated into the genome of the cell. Also, preferably, the operably linked unit results in stable incorporation. By stable incorporation it is meant that the presence of the operably linked unit is maintained for successive cell generations, preferably for at least 5 generations, and most preferably for at least 50 generations.

Incorporation may result in integration of a single operably linked unit or in the formation of conca emerε. By concatemer it is meant a sequence of DNA consisting of a series of unit lengths repeated in tandem. Concatemers may be very long in that the unit length sequence can be repeated over and over. Concatemers may be formed in head to tail, or tail to tail, or head to head orientation. The formation of concatenates in the genome can be monitored by cleavage with a panel of restriction endonucleases. If an enzyme is used which has a single site within the linear molecule, head to tail concatenates are seen as unit length DNA fragments. The extent of concatenation is estimated from the ratio of hybridization intensity of the unit length fragment to that of terminal DNA fragments resulting from cutting at genomic sites that are flanking the insertion site. Tail to tail and head to head concatenates and single unit insertions can be distinguished by their restriction patterns. In addition, integration may occur at only one chromosomal site or at multiple sites.

The .in vivo formed operably linked unit can be analyzed to confirm its structure. Restriction endonuclease analysis is used to obtain information regarding its gross structure. Enzymes are used that do not cleave within the introduced DNA molecules but do cleave in the flanking genomic DNA. The number of such DNA fragments that hybridize with a probe specific for the introduced DNA indicates the number of integration sites. Restriction endonuclease analysis is also used to obtain a preliminary analysis of the molecular structure of the integrated DNA. By carefully choosing the restriction enzymes for cleaving the DNA, a small junction DNA segment can be released in which removal of as few as about 50 nucleotides from the injected DNA molecule by a non-specific nuclease during concatenation is readily detectable by gel electrophoresis. Also, variations in the length of the junction fragment indicate incorporation of more than one processed monomer into the concatenate. A detailed analysis of the molecular structure is obtained with PCR amplication and sequencing. An upstream PCR primer and a downstream PCR nrimer are used. Assays for expression of the recombinant coding region can also be performed. Where multiple coding regions are introduced, each of the coding region products preferably is assayed. Assays include histochemical assays, immunohistochemical assays, enzymatic assays, protein purification, in situ hybridization methods in whole organisms, tissue sections, cell homogenates or single cells, RNA hybridization and RNAse protection assays. If the gene product of one of the coding regions is closely related to an endogenous gene product such that antibody cross-reactivity and in situ hybridization probe cross-reactivity are high, a determination of expression of such a coding sequence preferably is obtained by RNAse protection, specific antibody reaction, or PCR amplification. Where multiple regulatory regions are introduced e.g. , where the different regulatory regions direct expression in different tissues, expression of the coding region (or coding regions) is assayed in the expected range of tissues. Participation of the different regulatory regions in expression is assayed by PCR amplification using primers specific to each promoter in combination with primers specific to each coding sequence.

This invention also includes a method for changing the genotype of a non-human organism to produce an abnormal condition in the organism which simulates a condition sometimes occurring in nature. The term simulates a condition sometimes occurring in nature means that a condition is created in the organism which is similar to a condition that might occur in nature as a result of environmental events and/or other stimuli acting on the organism. By abnormal condition it is meant a condition which differs from the normal healthy condition of an organism. Such abnormal conditions include the acquisition of a characteristic which results in a diseased condition in the organism. Examples of a diseased condition include cancer, immune system deficiency, inflammatory disease, neurodegenerative disease, diabetes, thrombus formation and inherited metabolic disorders. An abnormal condition also includes a condition which increases the ability of an organism to withstand organ transplantation. Models can be produced for: cancer through the expression of viral proteins, activated oncogenes, and modulation of the expression of anti-oncogenes (Hanahan (1989) Science 246: 1265-1274; Bailleul et al. (1990) Cell: 62: 697-708; Haupt et al. (1991), Cell 65: 753-763; Lavigueur et al. (1989) Mol ■ Cell. Biol. 9: 3982-3991; Pattengale et al. (1989) Am. J. Path. 135: 39-61; Donehower et al. (1992) Nature 356: 215-221); development of the immune system through supplementation or elimination of molecular components of the immune response system (Hanahan (1989) Science 246: 1265-1274; Cosgrove et al. (1991) Cell 6 : 1051-1066; Bluethmann (1991) Experientia 47: 884-890; Koller et al.

(1990) Science 248: 1227-1230; Zijlstra et al. (1990) Nature 344: 742-746; Grusby et al. (1991) Science 253: 1417-1420; Reichman-Fried et al. (1990) PNAS USA 82: 2730-2734; Huesmann et al. (1991) Cell 6: 533-540; Robey et al . (1991) Cell 64: 99-107); organ transplantation through the expression of histocompatibility antigens on xenografts (Rocca et al .

(1991) Transplantation 52: 1062-1067; Auchincloss et al. (1990) Transplantation Proceedings 22: 1059-1060; Auchincloss et al. (1990) Transplantation Proceedings 22: 2335-2336; Nickerson and David, (1991) Transplantation Proceedings 23: 421-422); inflammatory disease through the production of a particular histocompatibility allele (Hammer et al. (1990) Cell 63: 1099-1112); neurodegenerative disease through the production of disease-state peptides in the brain (Hsiao and Prusiner (1991) Alzheimer's Disease and Associated Disorders 5: 155-162; Quon et al. (1991) Nature 352: 239-241); diabetes through the induction of autoimmune disease in the pancreas (Martin et al. (1990) New Biologist 2: 1101-1110); thrombus formation through the production of an inhibitor of plasminogen activator (Erickson et al. (1990) Nature 346, 74-76); and gene therapy through the correction of inherited metabolic disorders (Doetschman et al. (1987) Nature 330: 576-578; Thompson et al. (1989) Cell 56: 313-321).

This invention further includes a method for changing the genotype of an organism derived at least partially from a treated cell so as to obtain a desired characteristic, which may be predetermined, in the organism. Examples of a desired characteristic in an animal include enhanced milk production, disease resistance, growth enhancement, enhanced nutritional value and production of a desired protein. Examples of a desired characteristic in a plant include disease resistance, altered ripening characteristics, growth enhancement, enhanced nutritional value and production of a desired protein. Genes involved in disease resistance include gene products that counteract viral infections. (Muller and Brem (1991) Experienta 47: 923-934). Enhanced milk production is meant to include increased levels of milk, enhanced and/or novel nutritional value of milk, and milk containing various pharmaceutical products. Pharmaceutical products that can be produced in milk include tissue plasminogen activator and α-1-antitrypsin (Wright et al. (1991) Bio/technology 9_: 830-834; Ebert et al. (1991) Bio/technology 9: 835-843). Growth enhancement is meant to include faster growth, increased body size and increased litter size. Genes involved in growth enhancement include the gene for growth hormone. (Palmiter et al. (1982) Nature 300: 611-615); Palmiter et al. (1983) Science 222: 809-814). Enhanced nutritional value is meant to include greater amounts of a nutrient, novel nutrients, the absence of certain nutrients and leaner meat. Genes involved in enhanced nutritional value include the ski gene which results in leaner meat. (Colmenares and Stravnezer (1989) Cell 59: 293-303; Sutrave et al. (1990) Genes and Development 4: 1462-1472; Purεel et al. (1992) Theriogenology 37: 278). The term a desired protein means a protein that bestows a desired trait on the organism in which it is produced or a protein which when isolated from the organism is desirable for uses outside of the organism. The desired protein may be produced in a specific tissue, a subset of tissues or in a wide range of tissues, depending upon the use for which production of the protein is desired. Production of the protein also can be designed so that it is made at a desired time or times during the life of the organism. Examples of desired proteins include therapeutic proteins and proteins which correct an abnormal condition in the organism. Correction of an abnormal condition includes gene therapy.

Gene therapy, which has a wide variety of applications for mammals and in particular for humans, can be mediated by the introduction of the DNA encoding the therapeutic product by at least three different routes. One can introduce the DNA molecules into cells that are subsequently introduced into a person, one can package the DNA molecules within the genome of viruses that are subsequently used to infect a person, or one can deliver the DNA directly to cells within a person. Cells that can potentially be used are keratinocytes (Green, 1991, Sci. Am. 265, 96-102), lymphocytes Culver et al., 1991, PNAS USA 88, 3155-3159), bone marrow cells (Williams, 1990, Hum. Gene Ther . 1 , 229-239), endothelial cells (Nabel et al. 1992, Science 244, 1342-1344) or any other stem cell that has the desired properties of (1) taking up and expressing DNA molecules and either (2) persisting within the human body or differentiating into a persistent cell within the human body. Viruses that are current candidates for incorporation and introduction of therapeutic DNA molecules include retroviruses, and adenoviruses that can infect human cells (Culver et al., 1991, PNAS USA 88, 3155-3159; Wang et al, 1991, Adv. Exp. Med. Biol. 309, 61-66; Wilson et al. 1992, Science 244, 1344-1346; Rosenfeld et al . , 1992 68, 143-155). The field of direct DNA delivery is in its infancy but methods have been described using DNA/protein complexes, DNA/liposome complexes, and making use of the receptor-mediated endocytosiε pathway (Englehardt & Wilson, 1992, J. Pharm. Pharmacol. 44 165-167; Curiel et al . 1992, Pediatric Pulmonology 11_., 246; Curiel et al. , 1992, Am. J. Respir. Cell. Mol. Biol. 6 , 247-252; Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4434; Wu and Wu, 1988, J. Biol. Chem. 263, 14621-14624; Wu and Wu, 1988, Biochem. 2 , 887-892; Wu et al., 1991, J. Biol. Chem. 266, 14338-14342; Wilson et al., 1992, J. Biol. Chem. 267, 963-967; Bluestone, 1992, Bio/Technology 10, 132-136).

Obtaining the abnormal condition or obtaining the desired characteristic can be facilitated by, or even dependent upon, selection of a particular regulatory region. For example, the regulatory region may be preselected for its expected activity in a particular tissue or for its ability to be regulated by an event such as neoplastic transformation. Preselection will depend upon the characteristic or abnormal condition that is desired.

In addition to production of desired proteins in a recipient organism, or descendent from such recipient organism, this invention also provides for cultures of cell lines that can be the source for desired products as a result of containing the operably linked unit stably incorporated into the genome of the cells in the cell line.

This invention also includes breeding an organism, or an offspring of an organism, which was produced by the methods of this invention. Breeding is performed by normal and customary techniques.

This invention also includes a transgenic non-human organism having added a desired characteristic from having incorporated into the genome of at least some ceils an operably linked unit resulting from the method described above, a cell derived from a somatic cell from such a transgenic organism, and a cell line derived from such a somatic ceil.

This invention further includes a cell or a cell line having added a desired characteristic from having incorporated into its genome an operably linked regulatory region and coding region by any of the methods described above.

Examples Source of Materials

Restriction endonucleases are obtained from conventional commercial sources such as New England Biolabs (Beverly, MA) , Promega Biological Research Products (Madison, WI) and Stratagene (LaJolla, CA) . Radioactive materials are obtained from conventional commercial sources such as Dupont/NEN or Amersham. Custom-designed oligonucleotides for PCR are obtained from any of several commercial providers of such materials such as Bio-Synthesis Inc., Lewisville, TX. Animals suitable for transgenic experiments are obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY) and Harlan Sprague Dawley (Indianapolis, IN). Swiss Webster female mice are used for embryo retrieval and transfer. B6D2F., males are used for mating and vasectomized Swiss Webster studs are used to stimulate pseudopregnancy. Vasectomized mice and rats are obtained from the supplier.

Preparation of DNA for Transfec ions and Microinjections

DNA clones are cleaved with appropriate enzymes and the DNA fragments are electrophoresed on 1% agarose gels in TBE buffer. The DNA bands are visualized by staining with ethidium bromide, excised, and placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags, extracted with phenol-chloroform (1:1), and precipitated by two volumes of ethanol . The DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-D column. The column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer. The DNA solutions

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are passed through the column for three times to bind DNA to the column matrix. After one wash with 3 ml of low salt buffer, the DNA is eluted with 0.4 ml of high salt buffer and precipitated by two volumes of ethanol. DNA concentrations are measured by absorption at 260 nm in a UV spectrophotomer. For microinjection, the DNA concentration is.adjusted to 3 μg/ml in 5 mM tris, pH 7.4 and 0.1 mM EDTA.

Microinjection Procedures

The procedures for manipulation of the rodent embryo and for microinjection of DNA into the pronucleus of the zygote are described in detail in Hogan et al. , Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1986), the teachings of which are incorporated herein. In addition, microinjection of DNA molecules into the nucleus of a two-cell embryo is also used to produce transgenic animals. Brinster et al. (1985) Proc. Nat'l. Acad. Sci. , USA 82: 4438-4442. Similar methods are used to microinject DNA into eggs and embryos of other mammals. Hammer et al. (1985) Nature 315: 680-683; Murray et al. (1989) Reprod. Fert. Devi. 147-155; Pursel et al. (1989) Science 244: 1281-1288; Pursel et al. (1987) Vet. Immunol. Histopath. 17: 303-312; Rexroad et al. (1990) J. Reprod. Fert. 41 suppl. 119-124; Rexroad et al. (1989) Molec. Reprod. Devi. JL: 164-169; Simons et al. (1988) Biotechnology 6: 179-183; Vize et al. (1988) J. Cell Sci. 9 : 295-300; Wagner (1989) J. -Cell Biochem. 13B suppl. 164.

Microinjection procedures for fish and amphibian eggs are detailed in Houdebine and Chourrout (1991) Exnerientia 47: 891-897. Procedures for producing transgenic birds are detailed in Shu an (1991) Experientia 47: 897-905.

Other DNA Introduction Procedures

Procedures for introduction of DNA into plant protoplasts and other plant cells are detailed in Gasser and Fraley (1989) Science 244: 1293-1299; Goodman et ai. (1987) Science -21-

236: 48-54. Procedures for introduction of DNA into tissues of animals and into plants are described in Sanford et al . , US Patent # 4,945,050, July 30, 1990.

Procedures for Producing Transgenic Mice:

A. Embryo Recovery From Superovulated Females

Female mice six weeks of age, are induced to superovulate with a 5 IU injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma), followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG; Sigma) . Females are placed with males immediately after hCG injection. Twenty-one hours after hCG, the mated females are sacrificed by C0₂ asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma) . Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5% BSA (EBSS) in a 37.5°C incubator with a humidified atmosphere at 5% C0₂, 95% air until the time of injection.

B. Transfer of Embryos To Receptive Females Randomly cycling adult female mice are paired with vasectomized Swiss Webster males. Recipient females are mated at the same time as donor females. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with watchmakers forceps. Embryos to be transferred are placed in DPBS and in the tip of a transfer pipet (about 10-12 embryos). The pipet tip is inserted into the infundibulum and the embryos transferred. After the transfer, the incision is closed by two sutures. Procedures for Producing Transgenic Rats

The procedures for generating transgenic rats are similar to those for mice. Sprague Dawley rats are used for all procedures. Thirty day-old female rats receive a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female is placed with proven male. Also at this time, 40-80 day old females are placed in cages with vasectomized males. These will provide the recipient females for embryo transfer. The next morning females are checked for vaginal plugs. Females who have mated with vasectomized males are held aside until the time of transfer. Donor females that have mated are sacrificed (C0₂ asphyxiation) and their oviducts removed, placed in DPBS (Dulbecco's phosphate buffered saline) with 0.5% BSA and the embryos are removed with hyaluronidase (1 mg/ml). The embryos are then washed and placed in EBSS (Earle'ε balanced salt solution) containing 0.5% BSA in a 37.5°C incubator until the time of microinjection.

Once the embryos are injected, the live embryos are moved to DPBS for transfer into recipient females. The recipient females are anesthetized with ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip) . A dorsal midline incision is made through the skin and the ovary and the oviduct is exposed by an incision through the muscle layer directly over the ovary. The ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip of the transfer pipet is inserted into the infundibulum. Approximately 10-12 embryos are transferred into each rat oviduct through the infundibulum. The incision is then closed with sutures, and the recipient females are housed singly.

Identification of Transgenic Mice and Rats

Tail samples (1-2 cm) are removed from three week old animals. DNA is prepared and analyzed by both Southern blot and PCR to detect transgenic founder (F_Q) animals and the progeny (F, and F₂) . -23-

Production of Non-Rodent Transgenic Animals

Procedures for the production of non-rodent mammals and other iimals have been described by others. Hammer et al . (1985) Nature 315: 680-683; Murray et al. (1989) Reprod. Fert. Devi. 147-155; Pursel et al. (1989) Science 244: 1281-1288; Pursel et al. (1987) Vet. Immunol. Histopath. r7: 3Q3-312; Rexroad et al. (1990) J. Reprod. Fert. 41 suppl. 119-124; Rexroad et al. (1989) Molec. Reprod. Devi. 1 : 164-169; Simons et al. (1988) Biotechnology 6: 179-183; Vize et al. (1988) J. Cell Sci. 9_0: 295-300; Wagner (1989) J Cell. Biochem. 13B suppl■ 164; Houdebine and Chourrout (1991) Experientia 47: 891-897; Shuman (1991) Experientia 47: 897-905.

Identification of Other Transgenic Organisms

An organism is identified as a potential transgenic by taking a sample of the organism for DNA extraction and hybridization analysis with a probe complementary to the transgene of interest. Alternatively, DNA extracted from the organism can be subjected to PCR analysis using PCR primers complementary to the transgene of interest.

Evaluation of the Operably Linked Unit in Transgenic Non-Human Organisms

Evaluation of the gross structure of the operably linked unit in transgenic non-human organisms is carried out by conventional restriction endonuclease analysis as discussed above in order to reveal the number of integration sites.

A preliminary evaluation of the molecular structure of the junction of monomers of injected DNA molecules within concatenates is carried out by restriction endonuclease digestion. For example, in the case of concatenates formed after Smal cleavage of plasmid pCMVβ, cleavage with Xhol and Pvul in a head to tail concatenate yields a 500 base pair fragment. See Figure 5 which depicts plasmid pCMVβ with the relevant restriction endonuclease sites noted. Removal of as few as about 50 nucleotides from the injected DNA molecule by a non-specific nuclease during concatenation is readily detected by gel electrophoresis. Furthermore, in a long concatenate, variation in the length of the junction fragment indicates the incorporation of more than one processed monomer into the concatenate. Uniform junction fragment length of the expected size indicates that only non-processed monomers are present in the concatenate. Uniform junction fragment length of less than the expected size indicates that only one class of processed monomers is present in the concatenate.

A detailed evaluation of the molecular structure of the junction fragment is carried out through PCR amplification and sequencing. For example, see Figure 6, in which an upstream and a downstream PCR primer are used to amplify a junction fragment from the genome that is generated as a result of joining a CMV promoter region with a β-galactosidase coding region. The PCR product is then cleaved, for example, with Xhol and Pvul (see Figure 6), and is subcloned, for example, into a pBC plasmid vector (Stratagene, LaJolla, CA) for sequencing.

Similar strategies and techniques are employed to analyze the gross and detailed structures of DNA molecules introduced by the other methods described above.

Assay for Gene Activity in Transgenic Non-Human Animals

To evaluate embryonic expression of the transgene in a large number of individual animals, whole 14 day embryos are removed from transgenic mothers and fixed in 0.2% glutaraldehyde/1% formaldehyde following the procedure of Tan (1991) Devel. Biol. 146: 24-37. To evaluate expression of the transgene in adult tissues, individual tissues of interest are immersion-fixed following dissection from transgenic non-human animals. Alternatively, non-human animals are perfused with 4% paraformaldehyde following the procedure of Tan (1991). For example, expression of the β-galactosidase gene is analyzed by incubation with the chromogenic substrate X-gal which results in deposition of a blue reaction product (Tan, 1991).

Joining of Physically-Linked, But Not Operably Linked, Heteroloqous Promoter and Protein Coding Sequences

. This example is intended to show the viability of our technique for adding a desired trait. The plasmid, pCMVβ (Clontech, Palo Alto, CA) , contains the promoter region from the dominant immediate early gene from human cytomegaloviruε (1.9kb mRNA; Boshart et al. (1985), Cell 4.:521-530) functionally linked to the β-galactosidase gene from the E. coli lactose operon. The CMV promoter is expressed in a wide variety of tissues in transgenic animals (Schmidt et al . (1990), Mol. Cell. Biol. ] ):4406-4411; Furth et al . (1991), Nuc. Acids Res. 1?:6205-6208) . Thus, cells in a transgenic animal that express this construct are identified by assaying for β-galactosidase enzyme activity. A common and simple β-galactosidase assay employs the chromogenic substrate, X-gal, producing an intense blue color. Because of the ease of this assay, this DNA molecule is used to exemplify the degree of success of the process.

A site for the blunt end cutting restriction enzyme, Smal, is positioned such that it lies between the promoter and the initiator methionine codon of the β-galactosidase coding sequence. Cleavage at this Smal site inactivates the β-galactosidase gene by separating the promoter from the β-galactosidase coding sequence. Thus, transcription from the CMV promoter does not proceed through the β-galactosidaεe coding sequence. Smal-cleaved pCMVβ DNA, prepared using the methods described in this application, is injected into pronuclei of fertilized mouse eggs and further used to generate transgenic animals following the procedures described in this application.

The structure of the injected DNA molecules in the genomes of transgenic animals thus produced iε monitored by -26-

cleavage with a panel of restriction endonucleases. A Sail site is centrally located within the injected linear molecule (see Figure 5), and head to tail concatenates are therefore seen as unit length DNA fragments. The extent of concatenation is estimated from the ratio of hybridization intensity of the unit length fragment to that of terminal DNA fragments resulting from cutting at the genomic Sail sites flanking the insertion site. Formation of head to head or tail to tail concatenates is monitored by cleavage with EcoRI which cuts asymmetrically within the linear molecule that is originally injected. Single site insertions are revealed by the presence of only two EcoRI DNA fragments, neither of which is unit length. Head to tail concatenateε regenerate the functional expression cassette while other concatenates and single site insertions of either promoter or coding sequence fragments do not. To test for operable linkage of the promoter with the coding sequence, tissues from transgenic animals are assayed for β-galactosidase activity as described in Tan (1991).

In the above-example, a blunt-cutting restriction enzyme is used to sever the promoter sequence from the coding sequence. In other instances, a restriction endonuclease that leaves sticky ends are similarly used. The choice of enzyme is dictated by the exact structure of the DNA molecules used in the production of transgenic animals.

Joining of the -Tropomyosin Promoter Sequence With A Protein Coding Sequence

In other embodimentε of the invention, it is useful to introduce a promoter sequence on one DNA molecule and a coding sequence on a different DNA molecule. For example, the promoter region of the rat α-tropomyosin gene is included in a 4.2 kb BamHI to Apal DNA segment (Ruiz-Opazo et al. J. Biol. Chem. (1990) 265:9555-9562). See Figure 7 in which this 4.2 kb fragment with some of itε restriction endonuclease siteε iε depicted. The arrow in the figure -27-

indicates the site and direction for the initiation of mRNA transcription. The Apal site indicated in the figure separates the promoter region from the protein coding region. The Apal site was altered through conventional molecular genetic techniques and synthetic oligonucleotide linkers to generate a Sail site. The β-galactosidase coding region from the pCMVβ clone shown in Figure 5 is removed as an Xhol to Sail fragment. The four nucleotides of the 5' sticky end of the Sail site, 5'-TCGA-3', are complementary to the 5' sticky ends of the Xhol site, 3'-AGCT-5'. Thus, when these DNA fragments are co-injected into the pronucleus, the sticky end sequences promote association and operable linkage of the promoter sequences with the coding sequenceε. Aε a reεult of this association, the α-tropomyosin promoter εequences direct expression of the β-galactosidase coding sequences in the cells of the animal.

The α-tropomyosin promoter is expreεεed in a variety of tissues including striated muscle, smooth muscle, fibroblasts, and brain (Goodwin et al. (1991) J. Biol. Chem. 266: 8408-8415). The β-galactosidase protein iε readily assayed in cells and tissues by providing the recombinant cell or tissue with any of a variety of subεtrates for the enzyme. The preferable subεtrate is the compound X-gal, which will produce an intense blue reaction product in the presence of the enzyme. An example of the use of this asεay system in a transgenic animal system iε reported in Tan (1991). Becauεe cellular promoter εequenceε incorporated in a tranεgene conεtruct in tranεgenic animalε are usually active in the appropriate tisεueε, tiεεues that normally express the α-tropomyosin gene now expresε an easily asεayed gene product, making it εimpler to investigate the developmental expression of α-tropomyosin.

One useful application for a tranεgenic animal bearing the described α-tropomyosin/β-galactosidase tranεgene is a simple test to assay the effects of teratogenε, mutagenε, or carcinogens on tiεεue development. Becauεe development of the mammalian fetus iε highly ordered in time and space, even a subtle change in development manifested by a change in the pattern of β-galactosidase positive cells and tissues is readily observed.

Joining of the α-Tropomyosin Promoter Sequence with Several Oncogene Protein Coding Sequences

For some purposes, it is useful to express more than one coding sequence in the same cell under control of the same promoter sequence. An example of such a use is the production of tumorε in laboratory animalε for testing anti-neoplastic agents. For example, tumors are produced in transgenic animals through the expression of activated oncogenes (Cardiff et al. (1991), Am. J. Path. 139:495-501). In general, tumors are produced after a lag period of many months. Expression of two different activated oncogenes in the same transgeneic animal promotes more rapid tumor formation, making the experimental syεtem more useful. Usually the transgenic animal expresεing two different oncogeneε iε produced through mating of parents, each parent expresεing a single activated oncogene.

The current invention is useful for achieving expresεion of two different oncogeneε in the εame tiεsues of a given transgenic animal without the requirement of generating two different animal lines and then mating them. The potential for savings in time and expense in producing and using the transgenic animals described here is great as compared to the production of existing tranεgenic animals.

The α-tropomyosin promoter DNA fragment described above is used as the εeparate promoter DNA fragment. The oncogeneε used are ras and myc (Pattengale et al. (1989) Am. J. Path. 135: 39-61). The coding regions of these oncogeneε are contained on the DNA fragmentε εhown in Figure 8. The ras oncogene is excised as a Smal fragment and the myc oncogene is excised as an Xbal to EcoRI fragment. The blunt ends of the Smal cut DNA fragmentε are left untreated while the sticky ends of restriction enzyme cleaved DNA fragments are filled in with DNA polymerase (Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2d. ed.), Cold Spring Harbor Laboratory Press), such that the ends of the molecules^' are blunt. Thus, when a mixture of the DNA fragments containing the α-tropomyosin promoter, the ras oncogene, and the myc oncogene are injected into a fertilized egg, the transgenic animal produced expresses both oncogenes in the same tissueε under control of the same promoter. Expresεion of the two activated oncogenes in a given tisεue iε obεerved by the generation of tumorε in tranεgenic animalε as they age, as compared to control animals. Expresεion of the two activated oncogeneε in a given tissue of transgenic animals at early developmental times is assayed by _.in situ hybridization experiments on tisεue εection using probes specific for the two oncogenes (Niedobitek and Herbst (1991) Int. Rev. Expt. Pathol. 32: 1-56).

This invention is employed to provide an even simpler aεεay for activity of the α-tropomyoεin promoter in tiεεueε of the animal line in which tumor formation iε being monitored. This is accomplished by causing β-galactosidaεe expreεεion to be brought under control of the α-tropomyosin promoter in the same tissueε in which the oncogene εequenceε are controlled by the α-tropomyoεin promoter. Thiε is accomplished by including the above noted DNA fragment encoding the β-galactosidaεe coding εequence in the mixture of DNA molecules injected into the fertilized egg. The α-tropomyosin promoter thus is controlling expression of two oncogenes as well as the eaεily assayable β-galactosidase gene in a given set of tissues in a given line of transgenic animalε. The simple β-galactosidaεe assay takes the place of the more laborious and expensive in situ hybridization.

Knowing the tisεue diεtribution of α-tropomyoεin promoter activity in a given line of tranεgenic animals allows one to make a decision about the potential utility of a line of transgenic animals early in the lifetime of the animal. This knowledge allows the savingε of time and expense in keeping and analyzing animals that would subsequently be found to be inappropriate for the intended use.

A further extension of this application of the invention involves the use of one or more promoters in addition to the α-tropomyosin promoter in the mixture of DNA molecules injected into fertilized eggs. The spectrum of tissues in which the additional promoters are active similarly increases the spectrum of tissues in which tumors are produced, thereby making the transgenic animals more useful.

Joining of Multiple Regulatory Regions With A Coding Sequence For Gene Therapy

For purposes of gene therapy in humans, it is useful to ensure continuous expreεεion of a coding region through the joining of εeveral different regulatory regionε with the desired coding region. For example, the coding region for factor VIII (Kaufman (1991) Ann. Hematol. 63: 155-165), a required component of the blood clotting cascade, can be linked to more than one regulatory region. Also, the coding region for growth hormone (Hennighausen et al. (1982) Nucl. Acid. Res. 1 : 2677; Campbell et al. (1984) Nucl. Acid. Res. 12: 8685) can be linked to more than one regulatory region. The desirability for more than one regulatory region is that the expression of a given single regulatory region cannot be guaranteed in human tissue derived from a stem cell. By bringing the coding region for the desired therapeutic protein under control of more than one regulatory region, each with distinct but potentially overlapping expresεion patternε, the protein can be expreεsed throughout development of the tissue. With current technology, human keratinocytes can be cultured in vitro and grafted back onto the original donor or onto a matched recipient (Green (1991) Scientific American 265: 96-102). Through the methodε deεcribed above, the coding regionε for factor VIII or for growth hormone are iεolated and the regulatory regionε for human β actin (Leavitt et al. (1984) US Patent Application No. 650,958; publiεhed as EP 174,608 (860319)), human hypoxanthine-guanine phosphoribosyltransferase and 3-phosphoglycerate kinase (Johnεon and Friedman (1990) Gene 88: 207-213), and human keratin (Marchuk et al. (1984) Cell 39: 491-498) are isolated. These regions are then joined in i o within cultured human keratinocytes. When grafted back onto a human recipient, the therapeutic proteins are expresεed aε the keratinocytes grow and develop.

EQUIVALENTS

Those skilled in the art will be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein.

These and all other equivalents are intended to be encompassed by the following claims.

Claims

1. A method of treating cells to add a desired characteristic, comprising: introducing into a cell at least one regulatory region and at least one coding region that are free of operable linkage to each other when introduced, said regulatory region and said coding region being constructed and arranged such that they can join in vivo to form an operably linked unit aiding in obtaining said desired characteristic; and permitting said regulatory region and coding region to join in vivo.

2. The method of claim 1 wherein multiple regulatory regions, with at least two of the regulatory regions being different, are introduced into the cell.

3. The method of claim 1 wherein multiple coding regions, with at least two of the coding regions being different, are introduced into the cell.

4. The method of claim 1, further comprising: isolating said cell or a descendant cell from said cell that contains the operably linked unit.

5. The method of claim 1, further comprising: isolating said cell or a descendant cell from said cell that contains the operably linked unit as perεiεtent DNA.

6. The method of claim 1, further comprising: isolating said cell or a deεcendent cell from εaid cell that contains the operably linked unit incorporated into the genome of the cell.

7. The method of claim 6 wherein the descendent cell contains the regulatory region joined to the coding region, and wherein the cell is also substantially free of regulatory regions joined to regulatory regions and coding regions joined to coding regions.

8. The method of claim 6 wherein the operably linked unit is incorporated into the genome at multiple sites.

9. The method of claim 6 wherein the operably linked unit is stably incorporated into the genome.

10. The method of claim 1, further comprising: isolating said cell, or a descendent cell from said cell, that contains the operably linked unit as part of an autonomously replicating unit in the cell.

11. The method of claim 1, further comprising: culturing the treated cell, and isolating a cell line containing the operably linked unit stably incorporated into the genome of the cellε in the cell line.

12. The method of claim 1 wherein the cell that is treated is a non-human cell, and further comprising: allowing said cell or a descendent cell from said cell to subεtantially contribute to the development of a tranεgenic non-human organism containing the operably linked unit.

13. The method of claim 12 wherein the transgenic non-human organism has in substantially all of its cells the operably linked unit.

14. The method of claim 1 wherein the cell that is treated iε a non-human cell, and further compriεing: allowing said cell or a deεcendent cell from εaid cell to substantially contribute to the development of a chimeric non-human organism containing the operably linked unit.

15. The method of claim 1 wherein the cell that is treated is a stem cell.

16. The method of claim 1 wherein the operably linked unit is adapted for gene therapy.

17. The method of claim 1 wherein the cell that is treated is part of an animal.

18. The method of claim 15 further comprising: introducing the treated cell into an animal.

19. The method of claim 1 wherein the regulatory region and the coding region are introduced into a zygote.

20. The method of claim 1 wherein the regulatory region and the coding region are introduced into a cell of an early stage embryo.

21. The method of claim 19, further comprising: impregnating a receptive female with the zygote.

22. The method of claim 20, further comprising: impregnating a receptive female with the embryo.

23. The method of claim 21, further comprising: allowing the zygote to develop to term.

24. The method of claim 22, further comprising: allowing the embryo to develop to term.

25. The method of claim 1 wherein the coding region is normally regulated by a different regulatory region from the regulatory region introduced.

26. The method of claim 1 wherein before introduction into the cell, the downstream end of the regulatory region and the upstream end of the coding region have blunt DNA endε.

27. The method of claim 1 wherein before introduction into the cell, the downstream end of the regulatory region and the upstream end of the coding region have single stranded cohesive DNA ends that are complementary to each other.

28. The method of claim 27 wherein the single stranded cohesive DNA ends contain a high G+C content.

29. The method of claim 27 wherein the single stranded cohesive DNA end downεtream from the regulatory region iε compoεed of a firεt nucleotide and the εingle εtranded cohesive DNA end upstream from the coding region is compoεed of a εecond nucleotide that iε complementary to the first nucleotide.

30. The method of claim 1 wherein before introduction into the cell, the regulatory region has a sequence region at its downstream end that is homologous to a sequence region at the upstream end of the coding region.

31. In a method of producing a tranεgenic non-human organism, the improvement comprising: introducing into a non-human cell at least one regulatory region and at least one coding region that are free of operable linkage to each other when introduced, the regulatory region and the coding region being constructed and arranged such that they can join in vivo to form an operably linked unit.

32. The method of claim 12 or 31, wherein the transgenic non—human organism is a non-rodent animal.

33. The method of claim 12 or 31, wherein the transgenic non-human organism is a rodent.

34. A method for changing the genotype of an organism derived at least partially from a treated cell, so as to obtain a desired characteristic in said organism, comprising: introducing into a cell at least one regulatory region and at least one coding region that are free of operable linkage to each other when introduced, said regulatory region and said coding region being constructed and arranged εuch that they can join in vivo to form an operably linked unit aiding in obtaining εaid desired characteristic; permitting said regulatory region and coding region to join in vivo; isolating said cell or a descendent cell from said cell that contains said operably linked unit; and allowing εaid cell or εaid deεcendent cell to εubstantially contribute to the development of said transgenic non-human organism containing said operably linked unit.

35. The method of claim 34 wherein said desired characteristic is enhanced milk production.

36. The method of claim 34 wherein εaid deεired characteriεtic iε diεeaεe reεiεtance.

37. The method of claim 34 wherein εaid deεired characteriεtic iε growth enhancement.

38. The method of claim 34 wherein said desired characteristic is enhanced nutritional value.

39. The method of claim 34 wherein said desired characteristic is production of a desired protein.

40. The method of claim 39 wherein said protein is produced in desired tissues which are predetermined.

41. The method of claim 39 wherein said protein is produced at desired timeε.

42. A method for changing the genotype of a non-human organism to produce an abnormal condition in said organism which simulates a condition sometimes occurring in nature, comprising: introducing into a cell at least one regulatory region and at least one coding region that are free of operable linkage to each other when introduced, said regulatory region and said coding region being constructed and arranged such that they can join in vivo to form an operably linked unit aiding in obtaining said condition; permitting εaid regulatory region and coding region to join .in vivo; iεolating εaid cell or a deεcendent cell from εaid cell that containε said operably linked unit; and allowing said cell or said descendent cell to substantially contribute to the development of said transgenic non-human organism containing εaid operably linked unit.

43. A method for breeding an organiεm, or an offεpring of an organiεm, which organism or offspring was produced by a method comprising: introducing into a cell at least one regulatory region and at least one coding region that are free of -38-

operable linkage to each other when introduced, said regulatory region and said coding region being constructed and arranged such that they can join in. vivo to form an operably linked unit; permitting said regulatory region and coding region to join in vivo to impart a desired characteristic to said cell; isolating said cell or a descendent cell from said cell that contains said operably linked unit; allowing εaid cell or εaid deεcendent cell to εubstantially contribute to the development of said tranεgenic non-human organiεm containing εaid operably linked unit, εaid breeding method compriεing, mating εaid organiεm with another organism of an opposite mating type than said first mentioned organism.

44. A transgenic non-human organism, or an anceεtor of the organism, including in at least some of its cells a regulatory region and a coding region that were free of operable linkage when introduced into the organiεm, and wherein the regulatory region and the coding region were joined _.in vivo to form an operably linked unit.

45. A transgenic nonhuman organism as claimed in claim 44 wherein the operably linked unit aidε in imparting a deεired characteriεtic to said organism.

46. A transgenic nonhuman organism as claimed in claim 44 wherein the operably linked unit aids in imparting an abnormal condition to said organiεm.

47. A cell derived from the tranεgenic organiεm of claim 44, wherein the cell includeε said operably linked unit.

48. A cell, or a descendent of the cell, including in its genome a regulatory region and a coding region that were free of operable linkage when introduced into the cell, wherein the regulatory region and the coding region were joined in vivo to form an operably linked unit that aids in obtaining a desired characteristic.