JPH10509313A - Transgenic organisms and methods of using the same - Google Patents

Abstract

  (57) [Summary] Use of transgenic organisms (especially transgenic animals or plants) carrying positive and / or negative selectable markers in a variety of methods, including tissue / cell culture techniques, methods of producing monoclonal antibodies A method for selectively removing or deficient for a particular tissue / cell type; a method for screening compounds for pharmacological activity; Along with a method for measuring the effect on the properties of

Description

The present invention relates to transgenic organisms having cells with one or more selectable phenotypes and methods of using the same. Transgenic organisms, such as transgenic animals, have been known for many years. Although the term was sometimes applied to any organism containing exogenous DNA, the term "transgenic organism" is used herein in its more ordinary sense and refers to germline Eukaryotes (especially animals or plants, especially vertebrates such as mammals) containing heterologous chromosomal DNA therein and their progeny are indicated. This heterologous chromosomal DNA has a coding sequence hereinafter referred to as "transgene". Thus, any cell (or at least most of the cells) that a transgenic organism has-both somatic cells and germ cells-may contain one or more copies of this transgene. Transgenic organisms can be produced in many different ways. These methods are well documented in the prior art, and implementing these methods is part of the technical repertoire of those skilled in the art. Commonly used methodological approaches are described, for example, in Butterworth Heinemann, MA, USA, First Edition and "Hazeltine Edition" of "Transgenic Animals" (1991). In one known method, the transgene is inserted into fetal stem cells, which are then injected into a sterilized zygote, while only a few cells are present. The fetal stem cells thus treated begin to integrate into the zygote, and cells derived therefrom continue to differentiate into many or all of the different cell types of the animal. The cells also include those that contribute to the germ line, so that the progeny of this (chimeric) animal can be fully transgenic. Other methods include introducing the transgene into prokaryotes or into sterile or non-sterile eggs. It is also known in the art to treat cells or otherwise induce cells to express genes that impart any of a variety of selectable phenotypes on the cells. These genes are known as selectable markers. These are usually introduced into cells as part of a recombinant expression vector. The selectable phenotype conferred by the selectable marker can be classified as either positive or negative. A positive selectable phenotype is a phenotype that allows survival under certain circumstances that can kill (or at least inhibit or damage cell growth) cells that do not exhibit this positive selectable phenotype. is there. A negative selectable phenotype is an expression that results in the destruction (or inhibition or damage of cell growth) of this cell under specific circumstances that are relatively harmless to cells that do not display the negative selectable phenotype Type. A variety of selectable markers are available. Genes that can be widely applied as positive selectable markers include bacterial neomycin phosphotransferase (neo: Colbere-Garapin et al, 1981, 150: 1), hygromycin phosphotransferase (hph; Santerre et al. (Santerre et al.) et al) 1984 "Gene" 30: 147) and xanthine guanine phosphoribosyltransferase (gpt; Mulligan and Berg 1981 "PNAS" USA 78: 2072). Also used as positive selectable markers are herpes simplex virus type 1 thymidine kinase (HSV-1 TK; Wigler et al. (Wigler et al) 1977, Cell 11: 223), adenine phosphoribosyl Transferase (ARPT: Wigler et al. (Wigler et al) 1979, `` PNAS '' U.S.A. 76; 1373) and hypoxanthine phosphoribosyltransferase (HPRT; Jolly et al. (1983) `` PNAS '' U.S.A. 80; 477) . These latter markers must be used in cells with a particular mutant genotype (ie, one that results in a deficiency in the gene product on which selection is based). Also, some of the aforementioned genes result in a positive as well as a positive selectable phenotype. These include the HSV-1TK, APRT, HPRT and gpt genes. The enzymes encoded by these genes can catalyze the conversion of certain nucleoside or purine homologs to cytotoxic intermediates. For example, the nucleoside homolog ganciclovir (ganciclovir: GCV) is a good substrate for HSV-1 thymidine kinase, but a poor substrate for native thymidine kinase found in mammalian cells. There is only. As a result, GCV was able to select effective negative selections for cells expressing this HSV-1TK gene [St. Clair et al. 1987, “Antimicrob. Agents Chemother.” 31: 844]. Can be used to Xanthine guanine phosphoribosyltransferase, when expressed in wild-type cells [Besnard et al., 1987, "Mol. Cell. Biol." 7: 4139], has both positive and negative selection. Can be used. Selectable markers are commonly used in both prokaryotic and eukaryotic gene processing to allow cells that have undergone relatively rare genetic changes to be recovered from a mixed population. I have. For example, by using these markers in physical association with another gene that encodes a product of interest (eg, a therapeutic protein), cells inoculated with this other gene can be selected as described above. Can be selected with markers. For example, the neo gene has been used to monitor genetically modified cells taken from patient samples after gene therapy has been performed. It has also been proposed to use negative selectable markers as safety devices in gene therapy. Many gene therapies involve removing somatic cells from the patient, introducing a therapeutic gene into it, which repairs a biochemical lesion, and then reintroducing the cells back into the patient. For example. Genetically modified cells thus re-introduced may eventually prove to be detrimental to the patient's health (for example, if they are not immunocompatible). , Or when it turns out to be malignant), the subsequent selective removal of genetically modified cells (if necessary) by introducing a negative selectable marker with the therapeutic gene It is possible to do. Numerous vectors carrying positive or negative selectable markers have been produced and are readily available to those skilled in the art (for a review, see Miller (1992) Nature 357: 455-460). Others can be readily produced using standard gene cloning techniques. Transgenic organisms carrying selectable markers as transgenes are known in the art. In general, by introducing a selectable marker, cells that have also taken in the target gene can be collected, and the selectable marker is linked to the target gene. For example, many such transgenic organisms have been constructed in the course of research involving the introduction into germline cells with genetic information that disrupts normal phenotype (see WO / 91/13150). Also, transgenic organisms carrying selectable markers have been constructed in the course of constructing animals with specific genetic lesions. Here, the selectable marker has been inserted (usually by recombination of homology) into a particular gene, thereby inactivating the particular gene in the inserted state. The selectable phenotype conferred by the selectable marker is then used as a basis for the identification and recovery of cells that retain replication-inactivated replication of the gene. Examples of transgenic animals produced by this method are described in [Piedrahita et al., 1992, "PNAS" 89, pp. 4471-4475]. In vitro tissue / cell culture methods are fundamental to pharmaceutical, clinical, agricultural and industrial research. For example, tissue / cell culture methods have been used in the pharmaceutical industry for the preparation of pharmaceuticals (eg, therapeutically active protein products) or for measuring or screening drug potential. However, these in vitro cell / tissue culture techniques are slow, labor intensive, and expensive. One important problem is infection during culture (usually caused by microbial contamination by bacteria, yeasts and / or fungi). This is usually addressed by using various antibiotics, which are added into the medium to eliminate and reduce the growth of contaminants. However, the use of antibiotics does not completely eliminate the risk of infection, especially from contamination by yeasts and / or fungi, many of which are resistant to commonly used antibiotics. Another important problem arises from the need to culture a single tissue or cell type. In vitro growth from a single cell can be difficult (often requiring the use of a mixture of feeder cells and / or growth factors and other adjuncts), and thus a homogeneous in vitro Populations are not readily available. Therefore, there is a need for a convenient source of all types of cells / tissues, for primary culture or for other purposes. It has now been found that transgenic organisms carrying positive and / or negative selectable markers have previously unrecognized utility in cell culture technology. The transgenic organisms provided by the present invention constitute a very convenient source of material, particularly for the isolation, identification, culture and analysis of cells from any tissue of the organism's body. Tissues isolated from the transgenic organisms of the present invention can be particularly easily grown in vitro (even as a homogeneous population of a particular cell / tissue type), allowing for pharmacological measurements, synthesis of tissue grafts, It can be used for a wide range of applications, including drug delivery and protein production. According to one embodiment of the present invention there is provided a transgenic eukaryote, wherein the cells of the transgenic eukaryote encode a transgene encoding a positive selectable marker and a negative selectable marker. Heterologous DNA having a transgene that Except for a selectable phenotype resulting from this transgene, the organism may be substantially normal, and these transgenes may be arranged such that they inactivate the gene in an inserted state. Not in. As used herein, the term "substantially normal" means that the organism is not a mutant that produces any significant property or trait for the wild type and / or exhibits normal tissue differentiation and development. Is shown. For example, if the transgene of the organism resides in a silent (ie, non-expressed) region of its genome, and / or the transgene replicates, segregates, organizes or fills chromosomes, or ( The organism may be substantially normal in the sense that it resides in a region of the genome that does not significantly disrupt its interaction with cellular components such as DNA binding proteins (including histones and regulators). . Defining a transgene that encodes both a positive and a negative selectable marker provides great flexibility during subsequent manipulation of cells derived from the transgenic organism in vitro. Furthermore, when the present invention is used to generate tissue grafts, certain types of cells can be isolated from the transgenic animals of the present invention by positive selection. The cells thus isolated can then be transplanted into a non-transgenic animal to determine whether the graft has a therapeutic effect. The graft can then be removed with a negative selection to provide a control to determine whether the graft has a direct therapeutic effect. In another aspect of the invention, there is provided a transgenic eukaryote, wherein the cells of the organism encode a transgene encoding a positive selectable marker and / or a transgene encoding a negative selectable marker. And the organism is substantially normal except for a selectable phenotype resulting from this transgene. Positive and negative selectable markers can be provided by a single transgene. This is because some markers (as described above) can be used as both positive and negative selectable markers (depending on the selection environment employed). The transgenic eukaryote of the invention is preferably an animal or a plant, for example a vertebrate (eg a mammal such as a rat, rabbit, pig or mouse). Preferably, the transgenic organism has a genotype that is substantially wild-type except for the presence of the heterologous DNA. Further, this portion of the heterologous DNA that is expressed in the cell may be comprised of a transgene encoding a positive selectable marker and / or a transgene encoding a negative selectable marker, and The gene is operably linked to one or more expression factors. The absence of expression of the gene sequence from any other transgenesis makes this suitable transgenic organism suitable for extensive experimental studies that require a valid wild-type genotype background. . At least one of the selectable markers is operably linked to one or more regulatable expression factor (eg, one or more tissue- or cell-specific expression factors). In such circumstances, it may be advantageous to specifically modulate each selectable marker, for example, to make each marker a different tissue- or cell-specific one or more trait-expressing factors. To limit the expression of the selectable marker to a selected set of cells or tissues, thus, for example, from a mixed primary cell culture, to a selected set of cells. Alternatively, the present invention can provide for the in vitro selective culture of tissue. Neither does it rely on the use of organisms: any transgenic procedure may be employed in practicing the present invention, and in most circumstances, the precise nature of the selectable marker used in the present invention is not critical. In general, any selectable marker gene can be used as long as it confer a positive or negative selectable phenotype on the cell, eg, a positive selectable marker is neomycin phospho Selectable from transferase, hygromycin phosphotransferase, xanthine guanine phosphoribosyltransferase, herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltransferase, and hypoxanthine phosphoribosyltransferase. Can be selected from, for example, herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltransferase, hygromycin phosphotransferase, and hypoxanthine phosphoribosyltransferase.These selectable markers can be included in any of a number of known vectors (eg, restriction endonucleases). Conveniently, such as by subcloning using nucleases, examples of which are described in, for example, [Molecular Cloning: A laboratory Manual / s econd Edition] Sambrook J. Fritsch and Maniatis T. (Maniatis T) edited by Cold Spring Harbor Laboratory Press, 1989]. Trait expression factors for use in the present invention are those which are capable of directing and / or regulating (at least under certain circumstances) the trait expression of a gene to which they are operably linked. It can take any form as long as it is present. The expression factor for use in the present invention may comprise transcription and / or translation factors and have regulatory sites such as promoters, ribosome binding sites, enhancers, activators and repressor (operator) sites. May be. Suitable expression factors include promoters selected from those suitable for a wide range of uses, examples of which are shown in Table 1. This table is not exhaustive, but also shows how the promoters can be placed in the methods of the invention described below. By way of example only, a trait-expressing factor for use in the present invention may comprise: (i) dopaminergic, serotonergic, GABA-, cholinergic or peptidergic neurons and subpopulations thereof; (ii) oligodendrocytes Cells, astrocytes and subpopulations thereof, (iii) endocrine glands, lung, muscle, gonads, gastrointestinal, skeletal tissue or a part or parts thereof; (iv) epithelium, fibroblasts, fat, mast, A promoter or enhancer that is specifically active in leaf or parenchymal cells, (v) a particular stage of embryogenesis, and (vi) components of the blood system (eg, T-lymphocytes, B-lymphocytes and macrophages). You can choose from. Alternatively, they may include: (i) neurotransmitter-specific receptors; (ii) ion channels; (iii) receptors involved in ion channel gating; and (iv) cytokines, growth factors and hormones: And / or an enhancer that transcribes the gene for transcription. Advantageously, at least one of the selectable markers can be expressed as a component. This ensures uniform expression of the selectable marker under all circumstances in each transgenic cell of the transgenic organism, and allows the transgenic organism to be used as a source organism for cell / tissue culture for general use. This is particularly useful where it has been done. Constitutive expression can be achieved, for example, by using a promoter that promotes expression of a "growth essential" gene. A "growth essential" gene is one that is expressed in all cell types. The products from which they are translated are required as part of the general cellular metabolism or cellular structure, so that "growth essential" genes are not specifically expressed in certain cells or tissue types. Therefore, a growth-essential gene promoter needs to be active in a wide range (and sometimes all) of cell types to ensure constitutive gene expression. Examples of constitutively expressed promoters useful in the present invention include, for histocompatibility, the complex H-2K ^b Class 1 promoter [Weiss et al., 1983, Nature 301, 671-674; Baldwin and Sharp, 1987, Mol. Cell Biol. 7, 305-313; Kimura et al., 1986, Cell 44. 261-272], which have generally been shown to express downstream coding sequences in cells when used as promoters in transgenes [Jat et al., "PNAS USA" 88, 5096-5100]. Another example is the viral SV40 early promoter. The promoter used in the present invention is not limited to a promoter derived from a mammalian cell, and may also include a promoter derived from birds and fish. In addition, promoters derived from viruses, some of which have biological activity in a wide range of mammalian, fish and bird cells, and other eukaryotic cells, can also be used in practicing the present invention. For example, retroviruses having an early or late promoter derived from Simian virus-40, or promoter and enhancer elements, which promote expression of the sequence under the influence of the retrovirus in a wide range of eukaryotic cells There are retroviral terminal repeat units (LTRs) that have the ability to These promoters, as well as supporting sequences such as enhancer elements and other regulatory elements, are well known to those skilled in the art [see, eg, "Molecular Cloning: A laboratory Manual / second Edition" Sambrook J. et al. Edited by Fritz and Maniatis T, 1989, Cold Spring Harbor Laboratory Press. " Further, the transgenic organism of the present invention may further contain a heterologous DNA having a reporter transgene, for example, 3-galactosidase or luciferase. The reporter transgene is itself operably linked to one or more expression factors that are subject to cell or tissue specific regulation. Such a reporter transgene facilitates subsequent analysis of cells / tissues cultured from the transgenic organism, and in particular, the response of one particular trait expresser or set of trait expressors (eg, Response to defects induced in a set of cells / tissues) can be monitored in vitro or in vivo. In another aspect of the present invention, there is provided a method of culturing cells and / or tissues in vitro, comprising the steps of (a) transfecting a cell containing genetic material that confers a selectable phenotype on the cell. Providing a transgenic animal or plant; (b) generating a primary culture from cells and / or tissues of the transgenic organism of step (a); and (c) being contained in the cells of the transgenic organism. Selectively growing said primary culture based on said selectable phenotype conferred by said genetic material. Preferably, the cell / tissue culture method of the present invention employs a transgenic organism having a selectable marker operably linked to one or more tissue or cell-specific trait-expressing factors. Whereby, in step (c), specific cells / tissue types are selectively grown based on tissue / cell-specific expression within said at least one selectable marker. . The preferred method of the invention finds use, for example, in selecting thyroid vesicle cells from primary (mixed cell) cultures. According to this method, a primary stromal cell population of the thyroid gland can be brought about in the absence of thyroid vesicle cells, useful for the study of thyroid biology, and a new therapeutic agent for the treatment of thyroid disease Can constitute a unique cell culture system useful in the development of Also, the cell / tissue culture method of the present invention can be carried out in step (c) to reduce and eliminate microbial contamination of the tissue culture, thereby having a common problem in cell culture systems, namely, Contamination of the culture (particularly contamination by fungi and yeast) can be reduced or eliminated. In addition, the transgenic organism of the present invention can be used as a source of lymphocytes in a method for producing a monoclonal antibody. Monoclonal antibodies are of fundamental importance in biotechnology. This preparation method comprises the steps of (a) immunizing an animal by injecting an antigen of interest, (b) removing a spleen from the animal, preparing lymphocytes from the spleen, and (c) Producing hybridomas by fusing with immortal cells (usually myeloma cells), (d) selectively growing the hybridomas, and (e) cloning the hybridomas to secrete the desired monoclonal antibody A series of steps to produce a clone. The step of selectively growing hybridomas (step (d) above) can usually be achieved in a myeloma fusion partner, based on the HPRT genotype, wherein the myeloma fusion partner comprises unfused myeloma cells. Inhibit growth in selective media containing hypoxanthine, aminopeterin and thymidine (HAT media). This limits the choice of fusion partner. Accordingly, the method for producing a monoclonal antibody specific for an antigen provided in another aspect of the present invention comprises the steps of: (a) transgenic having a lymphocyte containing genetic material that confers a selectable phenotype on the lymphocyte; Providing an organism (eg, rat, rabbit, pig or mouse); (b) immunizing a transgenic organism with said antigen; (c) removing lymphocytes from said transgenic organism; (d) said step (c). Producing hybridomas by fusing the lymphocytes with immortal cells (eg, tumor cells such as myeloma cells); and (e) conferred by the genetic material contained in the lymphocytes. And selectively culturing the hybridoma based on a selectable phenotype. The presence of a selectable marker in the lymphocyte preparation from the transgenic organism eliminates the need for, for example, the HPRT selection process and expands the repertoire of fusion partner cells available for hybridoma generation. The transgenic organisms of the present invention also find use in diseases or disorders involving cell loss. Many diseases and disorders are known to be associated with the loss of certain cells and / or tissues. For example, in neurodegenerative diseases such as Parkinson's disease, Huntington's disease and Alzheimer's disease, one or more subpopulations of neurotransmitter identifying cells are lost during the course of the disease. In Parkinson's disease, although this loss is primarily a loss of dopaminergic neurons in the substabsia a nigra region of the brain, other cell types are also reduced. In Huntington's disease, there is a more general loss of neurons, but in this case this loss is largely limited to the striatum. In Alzheimer's disease, acetylcholine, serotonin and noradrenaline, which contain neurons that protrude into the neocortex and the old cortex, are reduced. Other neurological diseases and disorders also result from degeneration of nerve cells; for example, demyelination that occurs in multiple sclerosis is due to destruction of oligodendrocytes in the brain. Human immunodeficiency virus (HIV) is known to invade cells that express the CD4 receptor, and infection of the cells appears to ultimately result in cell death. Loss of CD4 cells results in catastrophic inhibition of the entire immune system and death of infected individuals. The molecular / cellular basis of the disease caused by HIV is poorly understood. This is due, at least in part, to the lack of a model system to study the development of this disease, especially in vivo. The adoption of primate-infected SIV (simian immunodeficiency virus) has been considered as a paradigm, but SIV-infected monkeys do not have a fully developed AIDS. In many cases, monkeys show no symptoms. Another model that has been proposed to date has been HIV-infected chimpanzees. Apart from the possibility of ethical considerations, the development of AIDS-like symptoms in such models takes several years, significantly delaying research and developing effective treatments. Thus, the animal models of the various diseases and disorders discussed above are essential as subjects for potential drugs and as subjects in basic clinical research. The choice of these animal models is currently very limited due to the difficulties associated with selectively destroying specific cells and / or tissue types. Accordingly, in another aspect of the present invention, there is provided a method for selectively removing or reducing a particular tissue or cell type in an organism, the method comprising: (a) providing a tissue or cell type to be removed or reduced. Providing a transgenic organism having a negative selectable marker operably linked to an expression factor (eg, a promoter) specific for (b) administering a selection agent to the organism. Removing or reducing said tissue or cell type based on the expression of said negative selectable marker within said tissue or cell type. The selective agent can be administered by any route. If systemic administration is required, oral, parenteral or intravenous routes can be employed. If local administration is required (eg, if the tissue or cell type to be removed is restricted to a particular organ or area of the body), targeted injection, subcutaneous implantation (eg, slow release) Capsules) or catheter insertion. For example, tissue within a particular region of the brain can be specifically targeted by intracerebral injection. Diseases / disorders involving loss of cells associated with the disease, such as immunity (such as AIDS, Parkinson's disease and Alzheimer's disease), employing the methods of the present invention for selectively removing or reducing specific tissues or cell types. An in vivo model of a deficiency or neurodegenerative disease / disorder can be provided. Accordingly, in another aspect of the invention, there is provided a method of modeling cell / tissue loss or atrophy associated with a disease / disorder, the method comprising: (a) subjecting the disease / disorder to removal or atrophy associated with the disease. Providing a transgenic organism having a negative selectable marker operably linked to a trait-expressing factor (eg, a promoter) specific for the tissue or cell type being treated, and (b) providing a selection agent. Administering to the organism, removing or reducing the tissue or cell type based on the expression of the negative selectable marker in the tissue or cell type. The present invention also provides a method (eg, an in vitro method) for determining the effect of deficiency of a first set of cells of an organism on the properties of a second set of cells, the method comprising: a) providing a transgenic organism having a first negative selectable marker operably linked to a trait expressor specific for a first set of cells, wherein the transgenic organism comprises: (I) a positive selectable marker operably linked to a trait-expressing factor specific for the second set of cells, or (ii) the presence of the organism excluding the second set of cells. (B) having any of the second negative selectable marker operably linked to a transcript that directs the expression of said negative selectable marker in all cells; ) Administering a selective agent to said organism Thereby causing a defect in said first set of cells based on intracellular expression of said negative selectable marker; (c) removing cells from said organism: and (d) (i A) removing in step (c) based on intracellular expression of said positive selectable marker, or (ii) based on lack of intracellular expression of said negative selectable marker. And selectively culturing the second set of cells from the collected cells. In another aspect of the present invention, there is provided a method of screening a compound for pharmacological activity against a disease or disorder involving cell / tissue loss or atrophy, comprising: (a) (i) treating a disease / disorder Providing a transgenic organism having a negative selectable marker operably linked to a trait-expressing factor (eg, a promoter) specific to the tissue or cell type that has been subjected to the associated ablation or atrophy, and then ( ii) administering a selection agent to the organism to remove or reduce the tissue / or cell type based on the expression of the negative selectable marker in the tissue or cell type to obtain a test model; Providing a test model of said disease by steps; (b) administering said compound to be tested to said test model; Screening each compound based on the effect of the compound on the test model of step (a). The method of the present invention can be effectively applied to any disease or disorder associated with cell / tissue loss or atrophy. In particular, the method of the present invention has particular utility in terms of neurodegenerative or immunodeficient diseases: for example, (a) Parkinson's disease (tissues containing dopaminergic neurons in nigra substances / to be removed or deleted / Or cell types), (b) Huntington's disease (tissue or cell type containing sulcus nerve cells to be removed or deleted), (c) Alzheimer's disease (acetylcholine, serotonin and / or associated with neocortex or old cortex) Or a tissue / or cell type containing noradrenaline neurons to be removed or deleted), (d) multiple sclerosis (a tissue / or cell type containing oligodendrocytes of the brain to be removed or deleted), ( e) cell types containing immune diseases and CD3, CD4 and / or CD8 cells to be removed or deleted) and Containing beauty (f) A IDS and CD4 cells, there is a cell type to be eliminated or defective. In the case of the AIDS model, the method of the present invention provides that a negative selectable marker is linked to a CD4 cell-specific promoter (eg, the CD4 receptor promoter), thereby specifically deficient or deficient in CD4 cells. Can be used to remove. Thus, in vivo negative selection could generate an in vivo model of AIDS by modulating the proportion of cells that express CD4. In addition, if the transgenic animal model carries both positive and negative selectable markers, any residual CD4 expressing cells may be removed from the transgenic tissue of this animal model for further study. Later isolation can be achieved by positive selection in vitro. Examples of various promoters suitable for use in the methods of the invention described above are listed in Table 1, along with the diseases for which each promoter will find use. The invention is also directed to cell / tissue cultures derived from the transgenic organisms of the invention (or produced by the cell culture method of the invention), and to various therapeutic uses of the invention. Is what you do. Hereinafter, the present invention will be described in more detail with reference to specific examples. These examples are not intended to be considered limiting in any way. For example, the methods and techniques required to construct a plasmid vector, to produce the present invention, are well known to those skilled in the art. The following arrangements thus constructed represent examples of the myriad constructions that can be used to practice the invention. The present invention should not be configured to be limited to only these applications. A. Material i. Vectors "pBabeneo plasmid vector" Morgenstern, J. et al. P. And Rand, H., "Nucl. Acids Res." 18 (1990) 3587-3596 (plasmids are freely available). "PCI plasmid vector" Promega, Madison, USA, 2800 Woods Halo Road "CD2 plasmid vector" Blaese, M.S. R. Bethesda, USA, National Institutes of Health (plasmids are freely available). Mullen, CA, Killstrap M., Blaez, RM, "Proc. Natl. Acad. Sci. USA" 89 (1992) 33-37. Austin, E. A. And Huber, BE [Mol. Pharmacol. 43 (1993) pp. 380-387. Wallace, PM, Macmaster, J. F. Smith, V. F. Carr, D. E. , Center, PD. And Caesando, WL. "Cancer Res." 54 (1994) 2719-2723. "TG-TK α plasmid vector" Wallace, H .; King's Wildings, University of Edinburgh, UK (plasmids are freely available). Wallace, H. , Ledent, C.E. , Vassart, G., Bishop, J. O. And Alshawi, R .; "Endocrinology" 129 (1991) 3217-3226. "PPBS plasmid" Morgan "Nucleic Acids Research", 1992, 20, 1293-1 299. ii. Molecular Reagent Restriction Endonuclease Promege, 28,000 Woods Hollow Road, Madison, USA. "DNA modifying enzyme, ligase, CIP, T4 polymerase, etc." Promege, 28,000 Woods Hallow Road, Madison, USA. Agarose for electrophoresis Sigma Chemical Corporation, St. Louis, U.S.A. Polynucleotide kinase and buffer, Unit 12, 3397 American Drive, Mississauga, Ontario, Canada, "New England Biolabs Limited." Gene Construction (i) Thyroglobulin-Thymidine Kinase-Internal Ribosome Introduction Site-Neomycin Resistance (TG-TK-α-IRES-neo ^r ) Neomycin resistance gene (neo ^r ) Was digested with HindIII / ClaI from the pBabe Neo plasmid [Morgenstern and Land "Nucl. Acids Res." 18 (1990) 3587-3596] ^r Obtained by gel electrophoresis of the 1165 bp fragment containing the gene and recovery with the "Promega Wizard PCR Kit". A pPBS plasmid containing a poliovirus derived from an internal ribosome entry site sequence [Morgan "Nucl. Acids Res." 1992, 20, 1293-1299] was digested with HindIII / ClaI. However, this can be done simultaneously or sequentially. Because these restriction sites were too close to each other. To overcome this problem, the plasmid is first digested with HindIII and these plasmids are inserted by inserting a 200 base pair fragment of DNA containing HindIII restriction sites at the 5 'and 3' ends. Sites were separated. The pPBS plasmid could then be digested first with ClaI and then with HindIII. Terminal phosphate groups were removed from the HindIII / ClaI-cut pPBS vector using bovine intestinal phosphatase (CIP). The vector was gel purified using a 1% agarose gel, the band containing the DNA was excised and electrophoretically eluted. The neomycin gene was then ligated into the pPBS plasmid overnight at 15 ° C., and the ligation reaction was transformed into freshly produced MC1061 recipient cells. Positive colonies were identified by digestion of the prepared plasmid with HindIII / ClaI. This "neo ^r Gene and plasmid were detected by electrophoresis in plasmid preparations from positive colonies. Plasmids from positive colonies were then digested with HincII and SacI (both restriction enzymes leave digested DNA with blunt ends). The Sac I / Hinc II digest containing the IRES-neoR fragment thus obtained was run on a 1% electrophoresis gel, the appropriate sized band was excised, the DNA was electrophoretically eluted and precipitated with ethanol. Was. TG-TKα plasmid (available freely from the National Institutes of Health, Gene Bank Accession No. JO2224; Santelli et al., 1993) DNA was prepared using a small device "Promega Wizard" and digested with NarI. did. The ends of this plasmid were blunt-ended by using T4 polymerase at 37 ° C. for 1 hour and then removing the terminal phosphate groups using CIP. The CIP was inactivated by treating the DNA with phenol / chloroform and then precipitating in ethanol. The thus obtained plasmid was electrophoresed on a 1% agarose gel, and the DNA was recovered and ligated to the insert at a molar ratio of the plasmid to the insert of 1: 3. This ligation was cultured overnight at 15 ° C. and then used to transform recipient MC1061 cells. Positive colonies were selected by digesting the prepared plasmid with BamHI (this correct construction results in restriction fragments of size 3980, 1663, 3102 and 1039 base pairs). Linearization of this plasmid was achieved by digesting the prepared plasmid with the SalI restriction enzyme. This structure is shown in FIG. (ii) Cytomegalovirus-cytosine deaminase-sv40 promoter neomycin resistance (CMV-CD-SV40-neo ^r , Or CD2-NEO ^r p) The pCD2 plasmid (Muren et al., PNAS 89 (1992) pp. 33-37) is digested with EcoRI and EcoRV, and the digest is electrophoresed on a 1% agarose gel, where the cytosine deaminase gene, SV40 A 2.5 kb fragment containing the promoter and the neomycin resistance gene was recovered by electrophoretic elution followed by ethanol precipitation. The fragment was treated with polynucleotide kinase to ensure that a terminal phosphate group was present in the fragment. The pCI vector is digested with EcoRI and SmaI (restriction enzyme leaving DNA with blunt ends), the terminal phosphate groups are removed using CIP, the enzyme is inactivated with phenol / chloroform and then precipitated in ethanol. Was. This band was then gel purified and collected by electrophoretic elution. A ligation with a 3: 1 molar ratio of insert to vector was prepared and performed overnight at 15 ° C. This ligation mixture is used to transform freshly prepared MC1061 recipient cells and positive colonies are selected by digesting the prepared plasmid with EcoRI and HindIII, increasing the length of 1868 and 5062 base pairs, respectively. A restriction fragment was obtained. Linearization of this plasmid was achieved by digestion with BglII. This configuration is shown in FIG. C. Production of transgenic animals Established methods (Hogan, B., Constantine, F. and Lacey, E., 1986), Handling of mouse embryos-Manipulating the Mouse Embro-A Laboratory Transgenic rats were produced according to (Cold Manual), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Briefly, approximately 2 pl of plasmid was microinjected into the pronucleus of a crossbred Sprague-Dawley embryo at a concentration of 5 μg / ml. The fetuses were then transplanted into pseudopregnant recipient cells and the strains were isolated and established after identifying transgenic animals. The lines were maintained as transgenic hemizygotes by crossing the hemizygous females with non-transgenic males. D. Positive / negative selection of cells from transgenic animals in vitro i. Fibroblasts mature CD2 / neo ^r , TG / TK / neo ^r And fibroblast cultures from the lungs of control animals were produced and expanded by conventional methods (Freshney (1987), Alan, R. Ris, New York). Twenty-four hours after plating, Geneticin (400 μg / ml) was added to nuclear cultures generated from both types of transgenic and control rats and replaced with fresh medium every three days did. When necessary, the cells were subcultured (1: 3) to prevent them from beginning to fuse, again by the basic method (Freshney, 1987). Cell counts were performed manually in 20 randomly selected fields of view, and the values at each time point were summed after changing by subculture. As can be seen from Table 2, the control animals and the TG / TK / neo survived for 10 days or more ^r There were no fibroblasts from the transgenic animals. In the absence of genoticin addition, no change in cell viability was observed in any of the transgenic animals. The effect of 5-fluorocytosine (5-FC) was also measured. A concentration of 100 μg of 5-fluorocytosine had no effect on fibroblasts from control animals or TG / TK / neor transgenic animals. However, in cells derived from CD2 / neor transgenic animals, 94% of the initially plated cells died or failed after 10 days of culture in the presence of 5FC (trypan blue). It was so determined that it could not be excluded) (Table 2). In contrast, cultures from control rat cultures with and without 5FC, and cultures from control and CD2 / neor rats without 5FC. There was no significant difference in cell counts between (Table 2). Drug was added to the cultures on day two. Each value is relative to the number of cells found in the control culture without drug addition at various time points after plating, allowing the dilution resulting from the subculture. Indication is the average of three different measurements and all standard errors were less than 15% of this average. ii. Thyroid cells mature CD2 / neo ^r , TG / TK / neo ^r Thyroid cultures derived from the thyroid gland of control and control animals were produced according to conventional methods (Freshney, 1987). Twenty-four hours after plating, Geneticin (400 μg / ml) was added to each culture generated from both types of transgenic and control rats and replaced with fresh medium every three days. did. When necessary, cells were subcultured (1: 2) to prevent them from beginning to fuse. Cell counts were performed manually in 20 randomly selected fields of view and, after changing by subculture, the values at each time point were summed (Table 3). Ten days after the first Geneticin application, 10 μg / ml acycloguanosine (ACG, Sigma) was added to TG / TK / neo. ^r Added to thyroid cells generated in transgenic animals. Ten days later, cells in 20 randomly selected fields were counted again. Table 3 shows the results. In summary, cells from both types of transgenic animals survived treatment with geneticin, whereas control cells did not. TG / TK / neo ^r Cells from transgenic animals did not survive treatment with ACG, whereas cells from control animals did. These results are, respectively, TG / TK / neo ^r And CD2 / neo ^r This was expected in terms of specific and non-specific phenotypic expression of positive and negative selectable markers in transgenic animals. TG / TK / neo cultured without any drug added ^r Transgenic rat thyroid cells did not show any differences in their growth or viability when compared to control thyroid cell cultures (Table 3). Drug was added to the cultures on day two. Each value is relative to the number of cells found in the control culture without drug addition at various time points after plating, allowing the dilution resulting from the subculture. Indication is the average of three different measurements and all standard errors were less than 15% of this average. E. FIG. CD2-neo ^r Improving fertility in tissue culture using cells from transgenic animals CD2-neo in transgenic rats ^r Cultures of spinal cord cells derived from E. coli were grown in small flasks using existing established methods (Foster et al., 1990, "Eur. J. Neurosci." 3, 32-39). . At the beginning of the experimental period, unknown amounts of other conventional laboratory microbial sources were introduced into the flask in the area of 100 yeast spores. At the same time, Geneticin (1 mg / ml) was added. After this, handling of all media and additives to the flask was performed outside the sterile environment of the laminar flow cabinet. After 60 days of the first plating, no evidence of any form of contamination was apparent. Indeed, neuronal survival and development appeared to be undamaged compared to uninfected control spinal cord cells grown in the absence of geneticin (see Figure 3. On day 14, (A) control rat fetus and (B) CD2 / neo ^r 1 shows a 60 day phase contrast micrograph of an in vitro spinal cord culture from a transgenic rat fetus. This latter culture was intentionally infected with yeast and other microorganisms and simultaneously treated with genotisin. No infection could be detected after applying genoticin, while cells infected without genoticin were overwhelmed by microbial growth in 4 or 5 days. F. Ablation of Thyroid Follicle Cells In Vivo Mature female rats (250 g) were injected intraperitoneally with 50 mg of ACG per day for 5 days. Seven days after this last injection, the respective serum levels of T3 and T4 were measured (Amersham, UK) and in transgenic animals decreased from 0.76, 0.05 nM to less than 0.06 nM (T3), 58.2, A decrease (T4) from 3.2 nM to less than 2.5 nM (N = 6) was found. Administration of salt to the transgenic animals resulted in a small but not significant decrease in T4 to 0.68, 0.07 nM (n = 6). The thyroid gland of the transgenic rats treated with ACG for 12 days atrophy to 7% of its original weight. Histochemical analysis of these thyroid glands revealed that almost all follicular cells had been lost, leaving only non-follicular cells, possibly calcitocin-producing cells. Administration of low doses of ACG per day resulted in partial loss of T3 and T4. In most other tissues from transgenic animals, HSV-thymidine kinase activity (Brinster et al., 1981, Cell 27, pp. 223-231; Jamison et al., 1974, J. Gen. Virol. 24 481-492) did not express in detectable amounts. No histochemical evidence of cell loss was shown in the parathyroid, submandibular or adrenal glands, nor in the heart, liver or brain. In summary, both types of transgenic animals, or cells derived therefrom, were clearly normal as appropriate until ACG or 5FC was applied. Following this application, the sensitized cells were rapidly destroyed, both in vivo and in vitro. Furthermore, cells from both transgenic organisms were resistant to the cytotoxic effects of geneticin, while cells from non-transgenic controls were completely annihilated. Example 2: Proposal of a Protocol for the Production of Transgenic Mice Carrying Positive and Negative Selectable Markers Herpes Simplex Virus (HSV) Thymidine Kinase Gene (tk) (Operably Linked to a tk Promoter ) And the bacterial neomycin phosphotransferase (neo) gene (operably linked to the SV40 early promoter) are cloned into appropriate cloning sites of a plasmid vector. This plasmid vector is digested with restriction endonucleases and a fragment containing both the tk and neo selectable markers (along with the expression factor operably linked thereto) is placed on an agarose gel. To separate. The fragments separated on this gel were then purified and placed in sterile unicellular mouse egg male prokaryotes in TE buffer (10 mM Tris, pH 7.5, 0.2 mM EDTA) at 1-2 μg / Inject at a concentration containing ml of DNA. These eggs are from the CBA x C57BL / 10 cross. The eggs that survived the microinjection were then transferred to pseudopregnant females for development and delivery, as described, for example, in Wagner et al., 1981, "PNAS", 78, p. 5016. At 7-14 days of age, each child was analyzed to determine if the transgene was present. DNA was prepared from a portion of the tail by the method described in [Sambrook et al., 1989, "Molecular cloning" Cold Spring Harbor]. The presence of the neo and tk genes is determined by measuring with labeled tk and neo specific probes. These transgenic offspring thus identified are bred and the offspring are also analyzed and tested for Mendelian inheritance of the transgene. Example 3 Proposal of a Protocol for the Selective Culture of Mouse Thyroid Follicle Cells Transgenic mice are prepared as described in Example 1, except that the neo gene has a thioglobulin promoter (eg Christoph et al. (1989)). `` Molecular and Cellular endocrinology '' 64 (1) pages 5-18; Christoph et al. (1987) 19, `` Suppl. '' 17, pages 70-73; and Ludent et al. (1990) `` PNAS '' 87 (16) 61 76 (Described on page -6180). Transgenic mice were sacrificed, their thyroid tissue was removed, and primary cultures were prepared in the presence of antibiotic G418. This antibiotic kills cells that do not express the neo gene and results in selective culture in primary cultures of thyroid follicle cells (mixed cells). Example 4: Proposal of a Preparation Protocol for Monoclonal Antibodies The bacterial neomycin phosphotransferase (neo) gene (operably linked to the sv40 early promoter) was cloned into a suitable cloning site of a plasmid vector. Was. The plasmid is then digested with restriction endonucleases, the fragment containing the neo selectable marker is separated on an agarose gel, and the transgenic mouse carrying the neo transgene is then substantially as described in the Examples. And prepared. The control antigen, in which the monoclonal antibody is required, is purified and injected with the aid of Freund's into the transgenic mice prepared as described above. The mouse is then sacrificed, the mouse spleen is removed and placed in tissue culture. The spleen is minced to release lymphocytes, and the lymphocytes are separated by centrifugation. These lymphocytes are then mixed with a myeloma fusion partner in the presence of polyethylene glycol to cause fusion and generate hybridomas. Hybridomas are selected by supplementing the culture medium with the antibiotic G148 based on the presence of the neo selectable marker in mouse lymphocytes. These hybridomas are then cloned by limiting dilution and the relevant clones are identified by screening by a suitable binding assay. This myeloma cell line need not have a negative selectable marker (eg, HPRT-). In addition, the presence of G418 in this culture reduces and eliminates the risk of infection of the culture. Example 5: Proposal of a protocol for the preparation of a rat model of Parkinson's disease Herpes simplex virus (HSV) thymidine kinase gene (tk) can be engineered into a promoter that is active only in dopaminergic neurons in nigra material And cloning into the appropriate cloning site of the plasmid vector. This plasmid was digested with restriction endonuclease, the fragment containing the tk selectable marker was separated on an agarose gel, and the transgenic rat carrying the tk gene was substantially described in Example 1. Prepared as described above. The ganciclovir is then injected by injection into the nigra extracellular region of the brain of the transgenic rat to specifically eliminate or abolish dopaminergic neurons expressing the negative selectable tk marker, Thus, a model rat of Parkinson's disease was obtained. Example 6: Proposal of a protocol for preparing a rat model of Alzheimer's disease The herpes simplex virus (HSV) thymidine kinase gene (tk) was expressed in acetylcholine, serotonin and / or noradrenergic neurons associated with the old and neocortex. It is operably linked to a promoter that is only active and cloned into an appropriate cloning site of a plasmid vector. The plasmid is digested by restriction endonuclease, the fragment containing the selectable tk marker is separated on an agarose gel, and the transgenic rat carrying the tk transgene is substantially described in Example 1. Prepare as described. The ganciclovir is then administered by injection into the appropriate area of the brain of the transgenic rat, whereby acetylcholine, serotonin and serotonin and neocortical-associated cortical, which express the negative selectable tk marker. // Noradrenaline neurons were specifically removed or deleted, whereby an Alzheimer's disease model rat was obtained.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] November 8, 1996 [Correction contents]                               The scope of the claims 1. A tissue derived from a transgenic organism, wherein the tissue is a transgenic organism. Heterologous DNA having a transgene that encodes a negative selectable marker And wherein the tissue is selected from the transgene. Substantially normal except for possible phenotypes, and transplant therapy (eg, neurodegenerative disease / Tissue used to treat disorders). 2. A tissue derived from a transgenic organism, wherein the tissue is a transgenic organism. Heterologous DNA having a transgene encoding a positive selectable marker And wherein the tissue is selected from the transgene. Tissue that is substantially normal except for possible phenotypes and is used for treatment. 3. A tissue derived from a transgenic organism, wherein the tissue is a transgenic organism. Has a transgene encoding a negative selectable marker and a positive selectable marker Cells containing heterologous DNA having a transgene encoding a functional marker Tissue that has and is used for treatment. 4. Transgenic animals or plants, such as vertebrates (eg, rat, cormorant) A mammal such as a heron, a pig or a mouse). The organization according to one claim. 5. The tissue is substantially wild-type except for the presence of the heterologous DNA; Have a genotype and / or form in the tissue the heterologous DNA A transgene encoding a positive selectable marker (such as a transgene) And, optionally, a transgene encoding a negative selectable marker) Each transgene is associated with one or more Operably linked The tissue according to any one of claims 1 to 4, wherein 6. At least one of said selectable markers is adjustable one or Multiple expression factors, for example, one or more tissue- or cell-specific forms The method according to any one of claims 1 to 5, which is operably linked to the expression factor. An organization according to one claim. 7. Each selectable marker is specifically regulated and each marker is Linked to one or more tissue or cell-specific expression factors 7. The tissue of claim 6, wherein 8. Claims wherein at least one selectable marker is constitutively expressed. An organization according to any one of claims 1 to 7. 9. The heterologous DNA may further comprise a reporter transgene, for example, β-galactosida. 9. A method according to any one of claims 1 to 8, wherein said method comprises luciferase or luciferase. The organization described in the section. 10. The reporter transgene is provided for regulation of tissue or cell specificity. Operably linked to one or more expression factors. Item 10. The tissue according to Item 9. 11. (A) the positive selectable marker is neomycin phosphotransf Enzyme, hygromycin phosphotransferase, xanthine guanine Phosphoribosyltransferase, herpes simplex virus type 1 thymidine quina , Adenine phosphoribosyltransferase and hypoxanthine Selected from phosphoribosyltransferases and / or Negative selectable markers are herpes simplex virus type 1 thymidine kinase, a Denin phosphoryl Bosyltransferase, hygromycin phosphotransferase and And hypoxanthine phosphoribosyltransferase, And / or (B) the trait expression factor is   (I) (i) dopaminergic, serotoninergic, GABAergic, choline -Active or peptide-active neurons and subpopulations thereof; (ii) oligodendrocytes Cells, astrocytes and subpopulations thereof, (iii) endocrine glands, lung, muscle, gonads, stomach Intestine, skeletal tissue or a part or parts thereof; (iv) epithelium, fibroblast Vesicles, fat, mast, mesenchymal or parenchymal cells; (v) specific stages of embryogenesis, And (vi) components of the blood system (eg, T-lymphocytes, B-lymphocytes, and macro- Promoters and / or enhancers that are specifically active in ー: Or (II) (i) neurotransmitter specific receptor; (ii) ion channel; ii) Receptors and (iv) sites involved in ion channel gating Promoters that transcribe genes for Caines, growth factors and hormones And / or enhancers The tissue according to any one of claims 1 to 10, wherein the tissue is selected from: 12. A method of culturing cells and / or tissue in vitro, comprising:   (A) Genetics with selectable markers that confer a selectable phenotype on cells Transgenic animal or plant having cells containing the substance, e.g. claims A transgenic animal having the tissue according to any one of claims 1 to 11. Or give plants;   (B) cells of the transgenic animal or plant of step (a) and / or Producing a primary culture from the tissue; and   (C) contained in the cells of the transgenic animal or plant Based on the selectable phenotype conferred by the genetic material, Each stage of selectively growing a culture,   The at least one selectable marker is a tissue or cell specific one or It is operably linked to a plurality of phenotypes, thereby providing a step ( In c), the specific cell / tissue type is converted to the at least one selectable marker. A method for selective growth based on tissue / cell-specific trait expression inside the car. 13. Producing a uniform population of a particular set of cells in the primary culture by step (c) 13. The method of claim 12, wherein 14． A method of culturing cells and / or tissue in vitro, comprising:   (A) Positive selectable marker that confers a positive selectable phenotype on cells A transgenic animal or plant having cells containing genetic material having For example, a transgene having a tissue according to any one of claims 1 to 11 Feeding the animal or plant;   (B) cells of the transgenic animal or plant of step (a) and / or Producing a primary culture from the tissue; and   (C) contained in the cells of the transgenic animal or plant Based on the selectable phenotype conferred by the genetic material, Each stage of selectively growing a culture,   Thereby, in step (c), the microorganisms of the tissue culture (for example, yeast and And fungi) to reduce or eliminate contamination. 15. Cultivated by the method according to any one of claims 12 to 14. Tissue or cells, wherein the tissue or cells are, for example, as tissue grafts, Use as analyte or source of desired protein in biochemical assays Tissue or cell that is to be treated. 16. The tissue or cell according to claim 15, which is used for treatment. 17． A method for producing a monoclonal antibody specific to an antigen,   (A) lymphocytes containing genetic material that confers a selectable phenotype on the lymphocytes A transgenic animal (e.g., any one of claims 1 to 11) A transgenic animal having the tissue described in the above section);   (B) immunizing said transgenic organism with said antigen;   (C) removing the lymphocytes from the transgenic animal;   (D) replacing the lymphocytes of step (c) with immortal cells (eg, tumor cells, eg, Producing a hybridoma by fusing with a myeloma cell; and   (E) the said gene provided by said genetic material contained in said lymphocytes Selectively culturing the hybridoma based on a selectable phenotype, The way you have. 18. An article for producing, for example for use in therapy, which can be produced by the method of claim 17. Clonal antibodies. 19. Selectively remove or delete specific tissues or cell types in an organism The method   (A) specific transduction for the tissue or cell type to be removed or deleted Negative selectable operably linked to current factor (eg, promoter) Having a functional marker, the tissue according to any one of claims 1 to 11, Providing a transgenic organism containing;   (B) administering said selective agent to said organism to produce said negative selectable This tissue or cell based on the expression of the marker within the tissue or cell type A method having each step of removing or deleting a mold. 20. Cell / tissue loss or atrophy (eg, immunodegenerative or neurodegenerative disease / disorder) 20. The method according to claim 19 for modeling harm).   If the tissue or cell type to be removed or deleted is a disease / disorder related removal A method that is a tissue or cell type that has been exposed or atrophyed. 21. Claims 1 to 11 for use in a method according to claim 19 or 20. A transgenic organism having a tissue according to any one of the preceding claims. 22. A specific cell / tissue produced by the method of claim 19 or 20. Organisms in which is specifically removed or deleted (eg, vertebrates such as mammals). 23. Models of cell / tissue loss or atrophy associated with disease / disorder, eg, immunity 23. The organism of claim 22, which is a model for a degenerative disease / disorder. 24. Cell / tissue loss or atrophy (eg, neuronal loss and / or Compounds for pharmacological activity against diseases or disorders including atrophy) Is a method of   (A) providing a test model of the disease / disorder according to the method of claim 20;   (B) administering to the test model of step (a) the compound to be tested;   (C) determining the compound to be tested based on the effect of the compound on the test model; And screening. 25. The disease / disorder is:   (A) Including Parkinson's disease and dopaminergic neurons in nigra substances A tissue / or cell type to be removed, deleted or deleted;   (B) Elimination containing Huntington's chorea and wrinkle neurons Or a tissue or cell type to be deleted;   (C) Alzheimer's disease and acetylcholine associated with neocortex or old cortex Ablation containing serotonin and / or noradrenaline neurons Tissue or cell type to be deleted   (D) Multiple sclerosis and removal or deficiency containing oligodendrocytes of the brain Tissue / or cell type to be treated;   (E) containing immune disorders and CD3, CD4 and / or CD8 cells The cell type to be removed or deleted; and   (F) a cell containing AIDS and CD4 cells to be removed or deleted; 25. The method of claim 20 or 24, wherein the method is of the vesicular type. 26. Deletion of a first set of cells of an organism results in a property of a second set of cells A method of measuring the effect (eg an in vitro method),   (A) operably linked to a trait-expressing factor specific to a first set of cells; 12. The method of any of claims 1 to 11, wherein the first negative selectable marker is Providing a transgenic organism having a tissue according to any one of the preceding claims. (I) operably linked to a trait-expressing factor specific for the second set of cells. A positive selectable marker that has been ligated; and (ii) the exception of a second set of cells, Transduction leading to expression of said negative selectable marker in all cells of the organism With either a second negative selectable marker linked to the current factor Yes;   (B) administering a selective agent to the organism, thereby predisposing the first set of cells into the organism; Caused a defect based on the intracellular expression of a negative selectable marker ;   (C) removing cells from the organism: and   (D) (i) based on the intracellular expression of the positive selectable marker Or (ii) lack of intracellular expression of said negative selectable marker. Selectively selecting said second set of cells from the cells removed in step (c) A method comprising the steps of:

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 39/395 ABB A61K 39/395 ABBN 48/00 48/00 ABA ABA C07K 16/00 C07K 16/00 C12N 5/10 C12P 21 / 08 C12P 21/08 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33/48 N 33/48 33/531 A 33/531 C12N 5/00 BC (81) Designated country EP ( AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, US, UZ, VN

Claims (1)

[Claims] 1. A transgenic eukaryote, the transgenic eukaryote The cell is a transgene encoding a positive selectable marker and a negative Comprising heterologous DNA having a transgene encoding a selectable marker Wherein the organism has a selectable phenotype, e.g., resulting from the transgene. A transgenic eukaryote that is substantially normal. 2. A transgenic eukaryote, the transgenic eukaryote The cell comprises a transgene encoding a positive selectable marker and / or Contains heterologous DNA with a transgene encoding a negative selectable marker. Wherein said organism excludes a selectable phenotype resulting from said transgene. A transgenic eukaryote that is substantially normal. 3. The transgenic eukaryote is an animal or a plant, for example, a vertebrate (E.g., a mammal such as a rat, rabbit, pig or mouse). Or the transgenic organism according to 2. 4. Wherein the transgenic organism has the exception that the heterologous DNA is present. Have a genotype that is substantially wild-type, and / or The portion of A that is expressed in the cell encodes a positive selectable marker Trans encoding a transgene and / or a negative selectable marker Genes, and each transgene has one or more A contract as claimed in any one of claims 1 to 3, which is operably connected to the child. The transgenic organism according to claim. 5. At least one of the selectable markers is an adjustable one or Multiple expression factors, for example, one or more tissue- or cell-specific forms 5. The method of claim 1, wherein the expression factor is operably linked to the expression factor. A transgenic organism according to one claim. 6. Each selectable marker is specifically regulated and each marker is Linked to one or more tissue or cell-specific expression factors The transgenic organism according to claim 5, wherein 7. Claims wherein at least one selectable marker is constitutively expressed. The transgenic organism according to any one of claims 1 to 6. 8. The heterologous DNA may further comprise a reporter transgene, such as 3-galactosida A method according to any one of claims 1 to 7, wherein the method comprises luciferase or luciferase. The transgenic organism according to Item. 9. The reporter transgene is subject to regulation of tissue or cell specificity Claims: 1. Operably linked to one or more expression factors. 9. The transgenic organism according to 8. 10. (A) the positive selectable marker is neomycin phosphotransf Enzyme, hygromycin phosphotransferase, xanthine guanine Phosphoribosyltransferase, herpes simplex virus type 1 thymidine quina , Adenine phosphoribosyltransferase and hypoxanthine Selected from phosphoribosyltransferases and / or Negative selectable markers are herpes simplex virus type 1 thymidine kinase, a Denin phosphoribosyltransferase, hygromycin phosphotran Spherase and hypoxanthine from phosphoribosyltransferase Selected and / or (B) the trait expression factor is   (I) (i) dopaminergic, serotoninergic, GABAergic, choline Active or peptide-active neurons and subpopulations thereof; (ii) oligodendrocytes Alveoli, astrocytes and subpopulations thereof, (iii) endocrine glands, lungs, muscles, gonads, gastrointestinal , Skeletal tissue or part or parts thereof; (iv) epithelium, fibroblast , Adipose, mast, mesenchymal or parenchymal tissue cells; (v) a particular stage of embryogenesis; And (vi) components of the blood system (eg, Specifically active on T-lymphocytes, B-lymphocytes and macrophages) A promoter and / or enhancer that is: or (II) (i) neurotransmitter specific receptor; (ii) ion channel; ii) Receptors and (iv) sites involved in ion channel gating Promoters that transcribe genes for Caines, growth factors and hormones And / or enhancers The transgenic device according to any one of claims 1 to 9, which is selected from the group consisting of: Enic creature. 11. The transgenic organism according to any one of claims 1 to 10. Tissue or cell derived or cultured from. 12. A method of culturing cells and / or tissue in vitro, comprising:   (A) Genes containing selectable markers that confer a selectable phenotype on cells A transgenic animal or plant having cells containing the substance, such as a transgenic animal or plant. A transgenic animal or plant according to any one of claims 10 to 10. Step;   (B) cells of the transgenic animal or plant of step (a) and / or Producing a primary culture from the tissue; and   (C) contained in the cells of the transgenic animal or plant Based on the selectable phenotype conferred by the genetic material, A method comprising the step of selectively growing a culture. 13. The at least one selectable marker is one that is tissue or cell specific. Or operably linked to multiple expression factors, thereby In floor (c), the specific cell / tissue type is selected from the at least one selectable type. Selectively grow based on tissue / cell-specific expression within the marker, eg 13. A method according to claim 12, wherein said method produces a uniform population of cells of a particular set of primary cultures. Method. 14． According to step (c), the microorganisms (eg yeasts and fungi) of the tissue culture 14. The method of claim 12 or claim 13 wherein the contamination is reduced or eliminated. 15. Cultivated by the method according to any one of claims 12 to 14. Tissue or cells, wherein the tissue or cells are, for example, as tissue grafts, Use as analyte or source of desired protein in biochemical assays Tissue or cell that is to be treated. 16. The tissue or cell according to claim 11 or 15, which is used for treatment. 17． A method for producing a monoclonal antibody specific to an antigen,   Has lymphocytes that contain genetic material that confers a selectable phenotype on the lymphocytes Transgenic animals (for example, as described in any one of claims 1 to 10) Transgenic animal);   (B) immunizing said transgenic organism with said antigen;   (C) removing the lymphocytes from the transgenic animal;   (D) replacing the lymphocytes of step (c) with immortal cells (such as myeloma cells); Producing hybridomas by fusion with such tumor cells; and   (E) the said gene provided by said genetic material contained in said lymphocytes A method for selectively culturing the hybridoma based on a selectable phenotype. 18. An article for manufacture, for example for use in therapy, which can be produced by the method of claim 17. Clonal antibodies. 19. Selectively remove or delete specific tissues or cell types in an organism The method   (A) specific transduction for the tissue or cell type to be removed or deleted Negative selectable operably linked to current factor (eg, promoter) A transgenic organism having a functional marker (for example, any of claims 1 to 10) A transgenic organism according to any one of the preceding claims);   (B) administering said selective agent to said organism to produce said negative selectable Based on the expression of the marker in the tissue or cell type A method comprising the steps of removing or deleting a tissue or cell type. 20. Cell / tissue loss or atrophy (eg, immunodegenerative or neurodegenerative disease / disorder) 20. The method according to claim 19 for modeling harm).   If the tissue or cell type to be removed or deleted is a disease / disorder related removal A method that is a tissue or cell type that has been exposed or atrophyed. 21. Claims 1 to 10 for use in a method according to claim 19 or 20. A transgenic organism according to any one of the preceding claims. 22. A specific cell / tissue produced by the method of claim 19 or 20. Organisms in which is specifically removed or deleted (eg, vertebrates such as mammals). 23. Models of cell / tissue loss or atrophy associated with disease / disorder, eg, immunity 23. The organism of claim 22, which is a model of a degenerative or neurodegenerative disease / disorder. 24. Diseases or disorders involving cell / tissue loss or atrophy (eg, neurons Compounds for pharmacological activity against loss and / or atrophy of Is a method of   (A) providing a test model of the disease according to the method of claim 20;   (B) administering to the test model of step (a) the compound to be tested;   (C) determining the compound to be tested based on the effect of the compound on the test model; And screening. 25. The disease is:   (A) Parkinson's disease and dopaminergic neurons in nigra substances A tissue / or cell type to be contained, removed or deleted;   (B) Huntington's disease and removal or deficiency containing sulcus neurons Tissue or cell type to be made;   (C) Alzheimer's disease and acetylcholine associated with neocortex or old cortex Ablation containing serotonin and / or noradrenaline neurons Tissue or cell type to be deleted   (D) Multiple sclerosis and removal or deficiency containing oligodendrocytes of the brain Tissue / or cell type to be treated;   (E) containing immune disorders and CD3, CD4 and / or CD8 cells The cell type to be removed or deleted; and   (F) a cell containing AIDS and CD4 cells to be removed or deleted; 25. The method of claim 20 or 24, wherein the method is of the vesicular type. 26. Defects in the first set of cells of the organism (eg, brain cells) are A method of measuring the effect on gender (eg an in vitro method),   (A) operably linked to a trait-expressing factor specific to a first set of cells; A transgenic organism having the first negative selectable marker ( For example, the organism according to any one of claims 1 to 10) is provided. So that the product is (i) operable with a trait-expressing factor specific for the second set of cells. A linked positive selectable marker, and (ii) before removing the second set of cells. Expression leading to expression of a negative selectable marker in all cells of the organism Having any of the second negative selectable markers linked to the factor R;   (B) administering a selective agent to the organism, thereby predisposing the first set of cells into the organism; Caused a defect based on the intracellular expression of a negative selectable marker ;   (C) removing cells from the organism: and (D) (i) based on the intracellular expression of said positive selectable marker, Or (ii) based on lack of intracellular expression of said negative selectable marker. And selectively removing said second set of cells from the cells removed in step (c). A method comprising the steps of culturing.