CZ130597A3 - Transgenic organisms and the use thereof - Google Patents

Abstract

Transgenic organisms (in particular, transgenic animals or plants) bearing positive and/or negative selectable markers are described along with their use in various methods including tissue/cell culture techniques, methods of making monoclonal antibodies, methods of selectively eliminating or depleting a particular tissue/cell type, methods of screening compounds for pharmacological activity and methods of determining the effect of a deficit in a first class of cells or the characteristics of a second class of cells in an organism.

Description

Technical field

The present invention relates to transgenic organisms having cells with one or more selectable phenotypes and their use.

BACKGROUND OF THE INVENTION

Transgenic organisms, including transgenic animals, have been known for many years. Although this term is occasionally used to refer to any oranges containing foreign DNA, the term transgenic organisms is used in its more usual sense to refer to eukaryotic organisms (and in particular animals and plants, and in particular vertebrates, ie animals) and their progeny containing heterologous chromosomal DNA. in the germ line. The heterologous chromosomal DNA includes coding sequences referred to herein as transgenic. Thus, each (or at least most) cells of the transgenic organism - both somatic and germinal - may contain one or more copies of the transgene (s).

Transgenic organisms can be produced by a number of different methods. Methods have previously been well described in the art and their implementation is part of the art. Commonly used methodological approaches are described, for example, in First and Haseltin (ed.), Transgenic Animals (1991) Butterworth-Heineman MA USA.

In one method, the transgene is inserted into embryonic stem cells, which are then injected into fertilized zygotes in a condition in which only a small number of cells are present. The processed embryonic stem cells are incorporated into the zygote and the cells derived therefrom differentiate into many or all different types. cells of the animal body. Such cells may also include those that form a germinative line, and the offspring of such (chimeric) animals may therefore be fully transgenic.

Another method requires the introduction of the transgene into the pronucleus or into a fertilized or unfertilized egg.

It is also known in the art that cells can be routinely processed or induced to express the gene (s), which then confer any of a wide variety of selectable phenotypes. Such genes are known as selectable markers. These are normally introduced into cells as part of a recombinant expression vector. The selectable phenotype conferred by the selectable marker can be both positive and negative.

A positive selectable phenotype is one that allows survival under certain conditions that should kill cells (or at least prevent or interfere with cell growth) that do not exhibit a positive selectable phenotype.

A negative selectable phenotype is one that leads to cell destruction (or prevents or interferes with cell growth) under certain conditions that are relatively harmless to cells that do not exhibit a negative selectable phenotype.

A wide variety of selectable markers are available. Genes that are widely used as positive selectable markers include bacterial neomycin phosphotransferase (neo; Colbere-Garapin et al., (1981) 150: 1), hygromycin phosphotransferase (hph; Santerre et al., (1984), Gene 30: 147 and xanthine guanine phosphoribosyl transferase (gpt; Mulligan and Berg (1981), PNAS, USA 78: 2072).

Also used as a positive selectable marker is herpes simplex virus type 1 thymidine kinase (HSV-1 TK; Wigler et al. (1977), Cell 11: 223), adenine phosphoribosyl transferase (APRT; Wigler et al., (1979), PNAS, USA 76: 1373) and hypoxanthine phosphoribosyltransferase (HPRT; Jolly et al., (1983), PNAS, USA 80: 477). These additional markers must be used in cells having a partially mutant genotype (one that leads to a deficiency of the gene product on which the selection is based).

Some of the aforementioned genes confer both a negative and a positive selectable phenotype. These include HSV-1 TK, APRT, HPRT and gpt genes. These genes encode enzymes that can catalyze the conversion of certain nucleosides or purine analogs to cytotoxic intermediates. For example, the nucleoside analogue ganciclovir (GCV) is a good substrate for HSV-1 thymidine kinase, but a poor substrate for native thymidine kinase mammalian cells. Thus, GCV can be used for effective negative selection against cells expressing the HSV-1 TK gene (St.Clair et al. (1987),

Antimicrob. Agents Chemother. 31: 844). Xantinguanine phosphoribosyl transferase can be used for both positive and negative selection when expressed in wild-type cells (Besnard et al., (1987), Mol. Cell Biol. 7: 4139).

Selectable markers are commonly used in genetic engineering of both prokaryotic and eukaryotic organisms to allow the recovery of those cells from a mixed population that have undergone relatively rare genetic change. For example, they can be used in physical association with another gene that encodes the desired product (for example, a therapeutic protein) to select cells into which that other gene has been introduced together with a selectable marker. For example, the neo gene was used to monitor genetically modified cells from patient samples after gene therapy.

The use of negative selectable markers as a safety device in gene therapy is also contemplated.

Many gene therapies require the removal of somatic cells from a patient, the introduction of a therapeutic gene (whose expression corrects the biochemical lesion), and then reintroduction of the cells back into the patient. Since reintroduction of genetically modified cells may ultimately be detrimental to patient health (for example, if they are immunologically incompatible or become malignant), a negative selectable marker may be introduced with the therapeutic gene to allow (if necessary) the subsequent selective elimination of the genetically modified cells .

A number of vectors carrying a positive or negative selectable marker have been made and are readily available to those skilled in the art (see, e.g., Miller (1992), Nature 357: 455 460). Others can be readily prepared using standard gene cloning techniques.

Transgenic organisms carrying a selectable marker as a transgene are known in the art. Generally, a selectable marker is introduced to allow for cells that also contain the desired gene to which the selectable marker is attached. For example, many such transgenic organisms have been constructed in a manner requiring the introduction of genetic information that disrupts normal development into the germ line (see WO / 91/13150).

Transgenic organisms carrying a selectable marker have also been constructed in the construction of an animal carrying a specific genetic lesion. Here, the selectable marker is inserted (usually by homologous recombination) into a single gene, which is thereby inactivated by this. The selectable phenotype conferred by the selectable marker is then used as the basis for identifying and obtaining cells carrying an insertionally inactivated copy of the gene. Examples of transgenic animals produced by such methods are described in Piedrahita et al., (1992), PNAS 89, pp. 4471-4475.

In vitro tissue / cell culture methods are essential for pharmaceutical, clinical, agricultural and industrial research. For example, tissue / cell culture methods are used in the pharmaceutical industry for the preparation of drugs (for example, therapeutically active protein products) and in screening or screening for potential drugs.

However, these in vitro cell / tissue culture techniques are slow, laborious and expensive. One important problem is culture infection (usually resulting from microbial contamination by bacteria, yeast and / or fungi). This is usually solved by using various antibiotics that are added to the culture medium to eliminate or reduce the growth of contaminants.

However, the use of antibiotics does not completely eliminate the risk of infection, especially that which originates from yeast or fungi (many of which are resistant to commonly used antibiotics).

Another important problem arises from the need to cultivate a single tissue or cell type. In vitro growth from a single cell may be difficult (often requiring the use of cell nutrition and / or a mixture of growth factors and other supplements) and therefore homogeneous in vitro populations cannot be readily obtained.

Therefore, there is a need for a suitable source of cells / tissues of all types for primary cultures and for other purposes.

SUMMARY OF THE INVENTION

It has now been discovered that transgenic organisms carrying a positive or negative selectable marker have previously unrecognized use in cell culture techniques.

The present invention provides transgenic organisms which, inter alia, constitute a very suitable source of material for isolating, identifying, culturing and analyzing cells from any tissue of the body of the organism. Tissues harvested from the transgenic organisms of the invention can be easily cultured (as a homogeneous population of individual cell / tissue types) in vitro and can be used in a wide variety of applications including pharmaceutical assays, tissue transplant synthesis, drug transfer, and protein production.

In one aspect of the present invention there is provided a transgenic eukaryotic organism having cells comprising heterologous DNA comprising a transgene encoding a positive selectable marker and a transgene encoding a negative selectable marker. In addition to selectable phenotypes arising from transgenes, the organism may be substantially normal, for example, the transgenes are not positioned to insertively inactivate the gene.

The term substantially normal as used herein means that the organism is not mutant for any significant property or feature relative to the wild type and / or exhibits normal tissue differentiation and development. For example, an organism may be substantially normal in the sense that the transgene is located in a silent (ie, unexpressed) region of the genome and / or in a region of the genome where the transgenes do not significantly interfere with chromosome replication, segregation, organization or condensation or interactions with cellular components such as DNA binding proteins (including histones and regulatory elements).

Providing transgenes encoding both positive and negative selectable markers provides greater flexibility during subsequent manipulation of cells derived from transgenic organisms in vitro. In addition, where the invention is used to generate tissue transplants, cells of individual types can be isolated from the transgenic animals of the invention by positive selection. Thus, isolated cells can then be transplanted into non-transgenic animals to determine if the transplant has any therapeutic effect. The transplant can then be removed by negative selection to provide control of whether the transplant has had a direct therapeutic effect.

In another aspect, the invention provides a transgenic eukaryotic organism having cells comprising heterologous DNA comprising a transgene encoding a positive selectable marker and / or a negative selectable marker, wherein the organism is substantially normal, except for a selectable phenotype derived from the transgene (s). Positive and negative selectable markers can be provided by a single transgene, as (as explained above) some markers can be used as both positive and negative selectable markers (depending on the selection conditions used).

The transgenic eukaryotic organisms of the invention are preferably animals or plants, for example vertebrates (i.e. mammals, e.g., rats, rabbits, pigs or mice).

The transgenic organism preferably has a genotype that is substantially wild type, except for the presence of heterologous DNA.

In addition, the portion of heterologous DNA that is expressed in cells may consist of a transgene encoding a positive selectable marker and / or a transgene encoding a negative selectable marker, wherein each transgene is operably linked to an expression element or elements. The absence of expression of any other transgenically derived genetic sequence makes this preferred transgenic organism suitable for a wide range of experimental research requiring a wild type genetic background.

At least one of the selectable markers may be operably linked to a controllable expression element or elements, for example a tissue or cell-specific expression element or elements. In such circumstances, each selectable marker is preferably differentially regulated, each marker being, for example, linked to a different tissue or cell specific element or elements. This allows the expression of the selectable marker to be limited to the selected class of cells or tissues, thus allowing selective cultivation of the selected class of cells or tissues in vitro from the primary cell culture mixture.

The present invention is not dependent on the use of transgenic organisms produced by any other method; any transgenic procedure may be used in an embodiment of the invention. In addition, in most circumstances, the exact properties of the selectable markers for use in the present invention are not important: in general, any selectable marker gene that confers a positive or negative selectable phenotype to cells can be used.

For example, the positive selectable marker may be selected from neomycin phosphotransferase, hygromycin phosphotransferase, xanthinguanine phosphoribosyl transferase, Herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltransferase, and hypoxantine phosphoribosyltransferase.

For example, the negative selectable marker may be Herpes simplex virus type 1 thymidine kinase, adenine phosphorosyltransferase, hygromycin phosphotransferase, and hypoxanthine phosphoribosyltransferase.

The selectable markers are suitably derived (ie, by subcloning using restriction endonucleases) from any of a number of known vectors, examples of which are described in Molecular Cloning: A Laboratory manual, second edition, issued by Sambrook J., Fritsch and Maniatis T., 1989, Cold Spring Harbor Laboratory Press).

Expression elements for use in the invention may take any form that allows (under at least some circumstances) control and / or control of the expression of genes to which they are operably linked. Expression elements for use in the invention may include transcriptional and / or translational elements, and include promoters, ribosome binding sites, enhancers, and regulatory sites including activator and repressor (operator) sites. Preferred expression elements include a wide variety of available promoters, examples of which are shown in Table 1. This table, which is not exhaustive, also shows its use in the methods of the invention described below.

For example, expression elements for use in the invention may be selected from: promoters and / or enhancers that are specifically active in: (i) dopaminergic, serotoninergic, GABAergic, cholinergic or peptidergic neurons and their subpopulations; (ii) oligodendrocytes, astrocytes and their subpopulations; (iii) endocrine glands, lungs, muscles, gonads, intestines, skeletal tissue or parts or portions thereof; (iv) epithelial, adipose, mast, mesenchymal, parenchymal cells and fibroblasts;

(v) individual stages of embryogenesis, and (vi) blood system components (i.e., T-lymphocytes, B-lymphocytes, and macrophages). Alternatively, they may be selected from promoters and / or enhancers that direct transcription of genes for: (i) neurotransmitter-specific receptors; (ii) ion channels; (iii) receptors involved in opening ion channels; (iv) cytokines, growth factors and hormones.

Preferably, at least one of the selectable markers may be constitutively expressed. This ensures uniform expression of the selectable marker in each transgenic cell of the transgenic organism under all conditions, which is particularly useful where the transgenic organism is for general use as a source for cell / tissue cultures.

Table 1

Promotor Type of tissue / cells Odl úaz Tyrosine hydroxylase Catecholaminergic neurons Alzheimerova ch. Parkinsonova ch. 1 TSH receptor Thyroid cells Hypothyroidism 2 BSF1 GABAergic neurons Epilepsy 3 Human dopamine 3-hydroxylase Noradrenaline neurons Alzheimerova ch. 4 Thyroglobulin Thyroid cells Hypothyroidism 5 Serotonin 2 receptor glial cells in serotoninergic projection areas neurodegeneative diseases 6 Mice interleukin 4 bone cells and hematopoietic system inflammatory processes 7 CD4 receptor CD4 expressing T cells AIDS 8 human choline acetyl- Acetylcholine neurons Alzheimerova ch. Motor diseases 9

neuron transferase

Links:

1: Stachowick et al. (1994) Molecular Brain Research

22 (1-4): 309-319.

2: Ikuyama and Nawata (1994)

Clinical Medecine 52 (4): 962-968

3: Motejlek et al. (1994) Journal of Biological

Chemistry 269 (21): 15265-15273

4: Hoyle et al. (1994), Journal of Neuroscience 14 (5th Pt

1): 2455-2463

5: Pichon et al. (1994) Biochemical Journal 298 (pt

3): 537-541

6: Ding et. al. (1993), Molecular Brain Research, 20 (3).

181-191

7: Bruhn et al. (1993), PNAS USA, 90 (20): 90 (20); 97079711

8: Nakavama et al. (1993), International Immunology,

5 (9): 817-824

Li et_al. (1993), Neurochemical Research 18 (3):

271-275

10: Sohanberg et al. (1991), PNAS 99 (2): 603-607

Constitutive expression can be achieved, for example, through the use of a promoter that directs expression of the house-keeping gene.

A house-keeping gene is a gene that is expressed in all cell types. Its translated products are required as part of the general cellular metabolism or cellular structure and, consequently, are not specifically expressed in individual cells or tissues. House keeping gene promoters are therefore active in a broad spectrum (and sometimes in all) cell types to ensure constitutive gene expression.

Examples of constitutively expressed promoter useful in the present invention is a promoter for Histocompatibility Complex H-2K ¹³ Class 1 (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 has been shown to express downstream coding sequences in cells generally when used as a promoter in a transgene (Jat et al., (1991), PNAS USA

88, 5096-500). Another example is the SV40 viral early promoter.

The promoters used in the present invention are not limited to those derived from mammalian cells, but may also include promoters derived from birds and fish. In addition, promoters derived from viruses, some of which have biological activity in a wide variety of mammalian, avian and fish cells, as in other eukaryotes, can also be used in embodiments of the invention. Examples are monkey virus 40 derived early and late promoters, or long repeating terminal sequences (LTRs) of retroviruses that contain a promoter as well as enhancer elements and have the effect of inducing expression of sequences in a wide range of eukaryotic cells. These promoters, together with support sequences such as enhancer elements and other regulatory elements, are well known to those skilled in the art (see Molecular Cloning: A laboratory Manual second edition, published by Sambrook J,

Fritsch and Maniatis T, 1989, Cold Spring Harbor Laboratory

Press).

The transgenic organisms of the invention may also contain heterologous DNA, which further comprises a reporter transgene, for example 3-galactosidase or luciferase. The reporter transgene may itself be operably linked to an expression element or elements that are subject to cellular or tissue-specific regulation.

Such a reporter transgene facilitates subsequent analysis of cells / tissues cultured from transgenic organisms, and in particular allows for a response (for example, induced deficiency in individual classes of cells / tissues) of a particular expression element or class of expression elements that is monitored in vitro or in vivo.

In another aspect, the invention provides a method for culturing cells and / or tissues in vitro, the method comprising the steps of: (a) providing a transgenic animal or plant having cells comprising genetic material that confers a selectable phenotype thereon; (b) generating a primary culture from the cells and / or tissues of the transgenic organism of step (a); and (c) selectively cultivating the primary culture based on the selectable phenotypes conferred by the genetic material contained in the cells of the transgenic organism.

Preferably, the cell / tissue culture method of the invention is based on the use of a transgenic organism having a selectable marker operably linked to a tissue or cell-specific expression element or elements by which, in step (c), a particular cell / tissue type is selectively cultured on a tissue or cell-specific expression of at least one said selectable marker.

This preferred method of the invention finds application, for example, in the selection of thyroid follicular cells from a primary (mixed) culture. This method can provide the primary stromal cell population of the thyroid gland in the absence of follicular cells and creates a unique cell culture system useful for studying thyroid biology and the development of new therapeutic drugs for the treatment of thyroid diseases.

The cell / tissue culture method of the invention may also be performed such that step (c) reduces or eliminates microbial contamination of the tissue culture and thus alleviates or eliminates the general problem of cell culture systems, see. culture contamination (especially fungi and yeasts).

The transgenic organisms of the invention can also be used as a source of lymphocytes in methods for producing monoclonal antibodies.

Monoclonal antibodies are essential in biotechnology. Their preparation comprises a sequence of steps comprising: (a) immunizing the animal with an injection of the desired antigen; (b) removing the spleen from the animal and preparing lymphocytes therefrom; (c) fusing lymphocytes with immortal (usually myeloma) cells to produce hybridomas; (d) selectively cultivating hybridomas; (e) cloning the hybridomas to produce a clone secreting the desired monoclonal antibody.

The hybridoma selective culture step (step (d), above) is usually accomplished based on the HPRT-genotype of a myeloma fusion partner that prevents unfused myeloma cells from culturing in a selective medium containing hypoxanthine, aminopeterine and thymidine (HAT medium). This limits the choice of fusion partners.

Thus, according to another aspect of the present invention, there is provided a method of producing a monoclonal antibody specific for an antigen, comprising the steps of: (a) providing a transgenic animal (e.g., rat, rabbit, pig or mouse) having lymphocytes containing genetic material conferring a selectable phenotype ; (b) immunizing the transgenic organism with an antigen; (c) collecting lymphocytes from the transgenic animal; (d) lymphocytes of step (c) with immortalized cells (e.g., tumor cells (i.e., myeloma cells)) to produce hybridomas based on a selectable phenotype conferred by genetic material contained in the lymphocytes.

The presence of a selectable marker in lymphocyte preparations from transgenic animals removes the requirement for HPRT selection and extends the repertoire of fusion partner cells that can be used in hybridoma formation.

The transgenic organisms of the present invention also find use in relation to diseases or disorders involving cell loss.

Many diseases and disorders associated with specific cell and / or tissue loss are known. For example, in neurodegenerative diseases such as Parkinson's disease, Huntington's chorea and Alzheimer's disease one or more sub-populations of the Eurotransmitter identifying cells are lost during the disease.

In Parkinson's disease, this loss mainly affects dopaminergic neurons in the substantia nigra region of the brain, although there is also a decrease in other cell types.

In Huntingt's chorea, this loss of neurons is more general, but in this case the deficit is significantly limited to the striatum.

In Alzheimer's disease there is a reduction in acetylcholine, serotonin and noradrenaline-containing neurons located in the neo- and paleocortex.

Other neurological diseases and disorders also result from nerve cell degeneration; the demyelination occurring in multiple sclerosis, for example, is due to the destruction of oligodendrocytes in the brain.

human immunodeficiency virus (HIV) is known to enter cells expressing the CD4 receptor, and infection of the cell eventually leads to cell death. Loss of CD4 cells causes catastrophic blocking of the entire immune system and death of the infected person.

The molecular / cellular basis of HIV-induced disease is little known. This is due, at least in part, to the lack of model systems for studying disease pathogenesis, particularly in vivo.

The use of SIV (simian immunodeficiency virus) infected oprimates was considered a paradigm, but SIV monkeys did not acquire fully developed AIDS. In many cases they showed no symptoms. Alternative designed models included HIV-infected chimpanzees. In addition to potential ethical considerations, the manifestation of AIDS-like symptoms on such a model may take several years, significantly impeding the research and development of effective therapy.

Thus, animal models of the various diseases and disorders mentioned above are important as test subjects for potential pharmaceutical and basic clinical research. The choice of these models is currently very limited due to the difficulties associated with the selective destruction of cell types and / or tissues.

Thus, according to another aspect of the present invention, there is provided a method of selectively eliminating or depleting certain tissues or cell types in an organism, comprising the steps of: (a) providing a transgenic organism having a negative selectable marker operably linked to a cell or tissue type specific expression element and (b) administering a selective agent of the organism to eliminate or deplete this type of tissue or cells by expressing a negative selectable marker.

The selective agent may be administered by any route. If systemic administration is desired, an oral, parenteral or intravenous route may be employed. If local administration is desired (for example, where tissue or cell type is restricted to a particular organ or body area), targeted injection, implantation (i.e., slow release capsules) or catheterization may be used. For example, tissue in certain brain regions may be specifically targeted by intracerebral injection.

The method of selectively eliminating or depleting a particular tissue or cell type of the invention makes it possible to provide in vivo models of diseases / disorders including disease-induced cell loss, for example immunodegenerative or neurodegenerative diseases / disorders (such as AIDS, Parkinson's and Alzheimer's disease).

In another aspect, the present invention provides a method for modeling cell losses or atrophy associated with diseases / disorders comprising the steps of: (a) providing a transgenic organism having a negative selectable marker operably linked to an expression element (ie, a promoter) specific for a cell or tissue type that is a disease associated with elimination or atrophy, (b) administering a selective agent of the organism to eliminate or deplete this type of tissue or cells by expressing a negative selectable marker.

The invention also provides a method (ie, an in vitro method) for determining the effect of a first class cell deficiency on second class cell characteristics of an organism, the method comprising the steps of: (a) providing a transgenic organism having a first negative selectable marker operably linked to a first class specific expression element cells and either: (i) a positive selectable marker operably linked to an expression element specific for a second class of cells, or (ii) a second negative selectable marker linked to an expression element that directs expression of a negative selectable marker in all cells of the organism except the second class of cells; (b) administering to the organism a selective agent for inducing a deficiency in the first class of cells by expressing a negative selectable marker; (c) collecting cells from the organism; and (d) selectively cultivating second class cells from the cells harvested in step (c) based on: (i) expressing a positive selectable marker, or (ii) lack of expression of a negative selectable marker.

Another aspect of the invention provides a method for investigating the pharmacological activity of compounds for diseases or disorders comprising cell / tissue loss or atrophy, comprising the steps of: (a) providing a disease test model by the steps of: (i) providing a transgenic organism having a negative selectable marker operably linked to an expression element (ie, a promoter) specific for a tissue or cell type that is a subject with a disease / disorder associated with elimination or atrophy, and then (ii) administering a selective agent to eliminate or deplete the tissue or cell type by expressing a negative selectable a marker to produce a test model; (b) administering the test compound to a test model; (c) examining the test compound based on its effect on the test model of step (a).

The methods of the invention may advantageously be applied to any disease or disorder that is associated with cell or tissue loss or atrophy. More specifically, the methods of the invention find particular utility with respect to neurodegenerative and immunodegenerative diseases and disorders, for example: (a) Parkinson's disease (eliminated or depleted tissue type or cell type includes dopaminergic neurons in substantia nigra); Huntington's chorea (eliminated or depleted tissue type or cell type includes nerve cells in the striatum); Alzheimer's disease (the eliminated or depleted tissue type or cell type includes acetylcholine, serotonin and / or noradrenaline neurons associated with neo- and paleocortex); (d) multiple sclerosis (the eliminated or depleted tissue type or cell type includes brain oligodendrocytes); (e) immune diseases (the eliminated or depleted tissue type or cell type includes CD3, CD4 and / or CD8 cells) and (f) AIDS (the eliminated or depleted tissue type or cell type includes CD4 cells).

In the case of the AIDS model, the method of the invention can be used to specifically deplete or eliminate CD4 cells by binding a negative selectable marker to a specific CD4 cell promoter (i.e., a CD4 receptor promoter). This will allow the generation of an AIDS model in vivo by regulating the number of cells expressing CD4 by negative selection in vivo.

In addition, if the transgenic animals carry both a positive and a negative selectable marker, any residual CD4 expressing cell can later be isolated from the transgenic tissue of an animal model by positive in vitro selection for further study. Examples of various promoters suitable for use in the methods of the invention described above are listed in Table 1, along with diseases in which each promoter can find utility.

The invention also relates to the cell / tissue cultures derived from the tanning organisms of the invention (or produced by the cell culture methods of the invention), as well as various therapeutic uses of the invention.

The invention will now be described in more detail by way of specific examples. These examples are not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

Methods and technologies required for construction of plasmid vectors, for example, for carrying out the invention are well known to those skilled in the art. The constructed sequences given below represent examples of a large number of constructs that can be used in embodiments of the invention. The invention should not be limited to the use of only these constructs.

A. Material

i. vectors pBabeneo plasmid vector pCI plasmid vector

CD2 plasmid vector

Morgenstern, JP & Land, H. Nucl. Acids Res. 18 (1990) 3587 ³⁵⁹⁶ (plasmid is freely available)

Promega, 2800 Woods Hollow Rd, Madison, United States

Blaese, M.R., NIH, Bethesda, USA (plasmid is freely available) Mullen, C.A., Kilstrup, Μ., Blaese, R.M. , Why. Nati. Acad. Sci. USA, 89 (1992) 33-37. Austin, E.A. & Huber, B.E. Mol. Pharmacol., 43 (1993) 380-387. Wallace, P.M., MacMaster, J.F. Smith, V.F., Kerr, D.E., Senter, P.D. & Cosand, W.L. Cancer Res. 54 (1994) 2719-2723.

TG-ΤΚα plasmid vector Wallace, H., Kings Buildings, University of Edinburgh, UK (plasmid is freely available), Wallace, H., Ledent, C.,

Vassart, G., Bishop, J.O. & Al Shhawi, R. Endocrinology, 129 (1991) 3217-3226.

pPBS plasmid Morgan, Nucleic Acids Research (1992), 20, pages 1293-1299 ii. agents for molecular biology

Restriction endonucleases

Promega, 28000 Woods, Hollow Rd, Madison, United States

DNA modifying enzymes ligase, CIP, T4 polymerase, etc.

Promega, 28000 Woods, Hollow Rd, Madison, United States

Agarose for electrophoresis

Sigma Chemical Co. St. Louis, USA

Polynucleotide kinase and buffers

New England Biolabs LTD.

Unit 12, Mississauga, Ontario, Canada

B. Genes Construction (i) Thyroglobulin thymidine kinase - internal ribosomal entry site - neomycin resistance (TG-TK-a-IRES-neo ^)

The neomycin resistance gene was obtained from the pBabe Neo plasmid (Morgenstern & Land, Nucl. Acids Res. 18 (1990) 3587-3596) by digesting with HindIII / Cla I and searching for the 1165 bp fragment containing the neo ^r gene by gel electrophoresis and the Promega Wizard PCR kit.

The pPBS plasmid (Morgan, Nucl. Acids Res. (1992) 20, 1293 1299) containing the polio-derived internal ribosomal entry site sequence was digested with HindIII / Cla I. However, this could not be done simultaneously or sequentially because of restriction sites she was too close together. To overcome this problem, the plasmid was first digested with HindIII and 200 b.p. a DNA fragment containing HindIII restriction sites at both the 5 'and 3' ends was inserted to separate the sites. The pPBS plasmid could then be digested first with Cla I and then with HindIII.

Terminal phosphate sequences were removed from the HindIII / Cla I truncated pPBS vector using calf intestinal phosphatase (CIP). The vector was gel purified using a 1% agarose gel and the DNA band was oxidized and electroeluted.

The neomycin gene was then ligated into pPBS plasmids overnight at 15 ° C and the reaction mixture transformed into freshly prepared MC1061 competent cells.

Positive colonies were identified by digestion of prepared HindiII / Cla I plasmids. The Neo gene and plasmid were detected electrophoretically in plasmid preparations from positive colonies. Plasmids from positive colonies were then digested with Hinc II and Sac I (both restriction enzymes leave blunt-ended digested DNA). The result of Sac I / Hinc II digestion containing the IRES-neo fragment was subjected to electrophoresis on a 1% gel and strips of good size were excised and the DNA was electroeluted and ethanol precipitated.

TG-TKα plasmid DNA (freely available from Genbank, NIH, USA accession number JO2224, Santelli et al., 1993) was prepared using Promega Wizard mini prep and digested with Nar I. The ends of the plasmids were blunted with T4 polymerase at 37 ° C for 1 hour followed by removal of the terminal phosphate groups using CIP. CIP was inactivated by phenol / chloroform DNA treatment followed by ethanol precipitation. The resulting plasmid was subjected to electrophoresis on a 1% agarose gel and the DNA was collected and ligated with inserts in a ratio of plasmids to insert of 1: 3.

The ligation mixture was incubated at 15 ° C overnight and then used to transform competent MC1061 cells. Positive colonies were selected by digesting the prepared BamH I plasmids (the correct product yielded restriction fragments of 3980, 3102 and

1039 b.p.).

The linearization of the plasmids was achieved by digesting the prepared Sal I plasmids with a restriction enzyme. The construction is shown in Figure 1.

(ii) Cytomegalovirus - cytosine deaminase - sv40 neomycin resistance promoter (CMV-CD-SV40-neo ^{: c} ', or CD2-neo ^{: c} ') pCD2 plasmid (Mullen et al., PNAS 89 (1992) 33-37) was digested with EcoR I and EcoR V and the digestion product was subjected to electrophoresis on a 1% agarose gel of which a 2.5 kb fragment containing the cytosine deaminase gene, SV40 promoter, and neomycin resistance gene was obtained by electroelution followed by ethanol precipitation.

To ensure that terminal phosphate groups are present in the fragment, it was treated with a polynucleotide kinase.

The pCI vector was digested with EcoR I and Srna I (blunt-ended restriction enzymes), and the terminal phosphate groups were removed using CIP and the enzyme was inactivated by phenol / chloroform followed by ethanol precipitation. The band was then gel purified and collected by electroelution.

The ligation was given to have a 3: 1 molar ratio of insert and vector and was performed at 15 ° C overnight. The ligation mixture was used to transform freshly prepared MC1061 competent cells and positive colonies were selected by digesting the prepared EcoR 1 and Hind III plasmids to give restriction fragments of 1868 b.p. and 5062 b.p., respectively. Linearization of the plasmids was achieved by digestion of Bgl I.

The construction is shown in Figure 2.

C. Production of transgenic animals

Transgenic rats were produced by established methods (Hogan, B., Constantini, F. & Lacy, E. (1986) Manipulating The Embryo Mouse - A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY). Briefly, approximately 2 µl of plasmids were microinjected at a concentration of 5 µg / ml into the Sprague-Dawley embryo cross-linked pronucleoles. The embryos were implanted in pseudopregnant recipients and after identification of the transgenic animals the lines were isolated and determined. The lines were maintained as hemizygots by mating hemizygotic females with non-transgenic males.

D. Positive / negative cell selection from transgenic animals in vitro.

i. Fibroblasts

Cultures of fibroblasts from adult CD2 / neo ^r , TG / TK / neo ^r and control animals were produced and propagated by standard methods (Freshney (1987), Alan R.Liss, New York). Twenty-four hours after plating, geneticin (400 µg / ml) was added to cultures originating from both types of transgenic rats and control rats, and fresh medium was replaced every three days. If desired, cells were subcultured (1: 3) to prevent them from becoming confluent, again, by basic culture methods (Freshney 1987). Cells were counted manually in 20 randomly selected fields and values for each time, with respect to changes caused by subculture, were collected. As seen in Table 2, no fibroblasts derived from control animals or TG / TK / neo ^r transgenic animals survived more than 10 days of treatment with geneticin. No change in cell survival from transgenic animals was observed under the baseline of addition of geneticin.

The effects of 5-fluorocytosine (5-FC) were also determined. 5-Fluorocytosine at a concentration of 100 µg / ml had no effect on fibroblasts derived from control animals or from TG / TK / neo ^r transgenic animals. However, in cells derived from CD? / Neo? Transgenic animals, 94% of the cells initially plated died or failed (as determined by their failure to trypan blue exclusion) after 10 days of culture in the presence of 5FC (Table 2). In contrast, no significant difference in cell numbers was found between cultures from control rats in the absence or presence of 5FC, or between controls and cultures from CD2 / neo ^r rats in the absence of 5FC added (Table 2).

Table 2

Survival of pulmonary fibroblasts derived from control and transgenic rats, and the effect of various drugs.

Days of cultivation

Genotype / drug 1 3 5 7 9 11 Control 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! TG / HM / neo ^r 98 97 98 95 95 96 CD2 / neo ^x ' 92 93 92 98 105 98 Control + geneticin 99 101 85 23 5 2 TG / TK / neo ^r + geneticin 97 105 108 101 ý · 105 111 CD2 / neo ^r + geneticin 91 94 91 93 107 105 Control + 5FC 101 105 98 97 93 96 TG / TK / neo ^r + 5FC 98 96 95 92 92 93 CD2 / neo ^{: t} + 5FC 94 5 3 3 4 4 Drugs were added in day 2 < into culture. ' Values are related

for the number of cells found in control cultures without drug addition and at different times after plating, and take into account the dilutions caused by passage. Images are the average of three separate determinations, standard errors are always less than 15% of the average.

ii. Thyroid cells

Cultures of adult thyroid cells of CD2 / neo ^r , TG / TK / neo ³⁷ and control animals were produced by routine methods (Freshney (1987)).

Twenty-four hours after plating, geneticin (400 pig / ml) was added to cultures originating from both types of transgenic rats and control rats, and fresh medium was replaced every three days. If desired, cells were subcultured (1: 2) to prevent them from becoming confluent.

Cells were counted manually in 20 randomly selected fields and values for each time, with respect to changes due to subculture, were collected (Table 3). 10 days after the initial application of geneticin, 10 µg / ml acycloguanosine (ACG, Sigma) was administered to thyroid cells originating from TG / TK / neo ³⁷ transgenic animals. Ten days later, cells were counted again in twenty randomly selected cells. The results are given in Table 3. To summarize, cells derived from both types of transgenic animals survived the geneticin treatment, while the control cells did not. Cells derived from TG / TK / neo ³⁷ transgenic animals did not survive ACG treatment, whereas cells derived from control animals did. Results were expected in terms of specific and non-specific expression of positive and negative selection markers in TG / TK / neo ¹⁷ and CD / neo ³⁷ transgenic animals, respectively.

TG / TK / neo ³⁷ thyroid cells of transgenic rats cultured in the absence of addition of any drug showed no differences in growth or survival compared to thyroid cell control cultures (Table 3).

Table 3

Survival of thyroid cells derived from control and transgenic rats, and effect of various drugs.

Days cultivation Genotype / drug 1 3 5 7 9 11 Control 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! 100 ALIGN! TG / HM / neo ^ 91 95 93 92 101 99 CD2 / neo ^r 99 103 102 97 89 91 Control + geneticin 95 91 85 56 9 4 TG / BP / neo ^r + geneticin 104 105 98 88 93 98 CD2 / neo ^x + geneticin 91 94 91 93 107 105 Control + ACG 94 97 98 91 92 102 TG / HM / neo ^r + ACG 98 38 12 10 8 7 CD2 / neo ^r + ACG 98 90 93 93 97 88

Drugs were added on day 2 to the culture. Values are based on the number of cells found in control cultures without drug addition, and at different times after plating, and take into account dilutions due to passage. Images are the average of three separate determinations, standard errors are always less than 15% of the average.

E. Increased sterility in tissue cultures using cells from CDZ-neo ¹⁷ transgenic animals

Spinal cord cell cultures derived from CD2-neo ^r transgenic rats were cultured in small vials using previously established methods (Foster et al., (1990) Eur. J. Neurosci. 3, 32-39). At the beginning of the experiment, 100 yeast spores and an unknown amount of other common laboratory contaminants were introduced into the vial. geneticin (1 mg / ml) was added at the same time.

Thereafter, all handling of the media and vial additives was performed outside the sterile environment of the laminar flow chamber.

Six days after the initial plating, no form of contamination was detectable. Survival and development of neural cells appeared to be intact compared to uninfected control spinal cells cultured in the absence of geneticin (see Fig. 3, which shows photomicrography with a phase contrast of 60 days in vitro cultured spinal cells from control rat embryos (A) and from CD2. / neo ^r transgenic rat embryos (B) 14 days of gestation Later cultures were thought to be infected with yeast and other microorganisms and simultaneously treated with geneticin After infection, no infection was detected, whereas in infected cultures without geneticin, microorganisms overgrown within 4 or 5 days.

F. Ablation of thyroid follicular cells in vivo

Adult female rats (250 g) were injected intraperltoneally with 50 mg of ACG daily for 5 days. Seven days after the last injection, serum levels of T ₃ and T ₄ (Amersham UK) were measured and found to decrease in transgenic animals from 0.76 0.05 to less than 0.06 nM (T ₃ ) and 58.2 3, respectively. 2 nM to less than 2.5 nM (T ₄ ) (N = 6). Administration of saline solution to transgenic animals resulted in a small but non-significant decrease in T ₄ from 0.68 0.07 nM (N = 6).

Thyroid glands of transgenic rats treated with ACG for 12 days decreased to 7% of the original weight. Histochemical analysis of these thyroid glands revealed an almost complete loss of follicular cells with the persistence of only non-follicular cells, probably calcitonin-producing cells. Administration of less ACG per day resulted in a partial decrease in T and T. In most other tissues from

In 4 transgenic animals, HSV activity was not thymidine kinase activity (Brinster et al., (1981) Cell 27, 223-231; Jamieson et al.

(1974) J. Gen. Virology 24, 481-492) expressed in a detectable amount. No histochemical evidence of cell loss was demonstrated in gl. parathyroidea, submaxillary or adrenal glands, not in the heart, kidneys or brain.

In conclusion, both types of transgenic animals, or cells from these, were apparently normal until either ACG or 5FC was administered, as appropriate. After such application, both in vivo and in vitro, the cells that were granted sensitivity were rapidly destroyed. In addition, cells from both transgenic animals were resistant to the cytotoxic effect of geneticin, while cells from non-transgenic controls were completely eradicated.

Example 2: Proposed protocol for the production of transgenic mice carrying both a positive and a negative selectable marker

The herpes simplex virus (HSV) thymidine kinase (tk) gene (operably linked to the tk promoter) and the neomycin phosphotransferase (neo) bacterial gene (operably linked to the SV40 early promoter) are cloned into the appropriate cloning sites of the plasmid vector,

The plasmid vector is digested with restriction endonucleases and the fragment containing both the tk and neo selectable marker (along with the expression elements bound thereto) is isolated on an agarose gel.

The gel-isolated fragment is then purified and injected into male pronuclei of fertilized unicellular mouse eggs at a concentration of 1-2 µg / ml DNA in TE buffer (10 mM Tris, pH 7.5,

0.2 mM EDTA). The eggs are derived from CBA x C57BL / 10 crossing.

The eggs that survive microinjection are then transferred to pseudopregnant females as described by Wagner et al., (1981)

PNAS 78, 5016, and are allowed to complete the development.

At 7-14 days, each offspring is analyzed for the presence of transgenes. DNA is prepared from the tail sample by the method described by Sambrook et al., (1989) Molecular Cloning, Cold Spring Harbor.

The presence of neo and tk genes is determined by probing with labeled tk and neo specific probes.

The transgenic progeny thus identified are crossed and their progeny are also analyzed to examine Mendelian transgene transfer.

Example 3: Proposed protocol for selective culture of murine thyroid follicular cells.

Transgenic mice are prepared as described in Example 1, except that the neo gene is placed under the control of a thyroglobulin promoter (as described by Christophe et al. (1989), Molecular and cellular endocrinology 64 (1) 5-18); Christophe et al. , (1987)

19, Suppl. 17, pp. 70-73; and Ledent et al., (1990), PNAS, 87 (16), pp. 6176-6180).

Transgenic mice are sacrificed and thyroid tissue is removed and is primarily cultured in the presence of antibiotic G418. This antibiotic kills cells that do not express the neo gene and results in selective culture within the primary (mixed) culture of thyroid follicular cells.

Example 4: Proposed protocol for preparation of monoclonal antibody

The bacterial neomycin transferase (neo) gene (operably linked to the SV40 early promoter) is cloned into a suitable cloning site of the plasmid vector. The plasmid is then digested with restriction endonucleases, and the fragment containing the neo selectable marker is isolated on an agarose gel, and transgenic mice carrying the neo transgene are then prepared essentially as described in Example 1.

The antigen against which the monoclonal antibody is desired is purified and injected into transgenic mice prepared as described above with Freund's adjuvant. Mice are then sacrificed and spleen is removed and placed in tissue culture fluid. The spleen is disrupted to release the lymphocytes and these are then isolated by centrifugation. The lymphocytes are then mixed with myeloma phage partners in the presence of polyethylene glycol to induce fusion and produce hybridomas.

Hybridomas are selected by supplementing the culture medium with antibiotic G418 based on the presence of a neo-selectable marker in mouse lymphocytes. Hybridomas are then cloned by limited dilution and relevant clones are identified by screening using appropriate binding assays.

The myeloma cell line need not have a negative selectable marker (i.e., HPRT). In addition, the presence of G418 in the culture medium reduces or eliminates the risk of culture infection.

Example 5: Proposed protocol for preparation of a rat model of Parkinson's disease

The herpes simplex virus (tk) thymidine kinase (HSV) gene is operably linked to a promoter that is only active in dopaminergic neurons in the substantia nigra and is cloned into the appropriate cloning sites of the plasmid vector.

The plasmid is digested with restriction endonuclease, and a fragment containing the tk selectable marker is isolated on an agarose gel, and transgenic rats carrying the tk transgene are then prepared essentially as described in Example 1.

Ganciclovir is then injected into the substantia nigra regions of the brain of transgenic rats to specifically eliminate or deplete dopaminergic neurons expressing a negative selectable tk marker to provide a rat model of Parkinson's disease.

Example 6: Proposed protocol for the preparation of a rat model of Alzheimer's disease

The herpes simplex virus (tk) thymidine kinase (HSV) gene is operably linked to a promoter that is only active in acetylcholine, serotonin and / or noradrenaline neurons associated with neo- and paleocortex is cloned into a suitable cloning site of the plasmid vector.

The plasmid is digested with restriction endonuclease, and a fragment containing the tk selectable marker is isolated on an agarose gel, and transgenic rats carrying the tk transgene are then prepared essentially as described in Example 1.

Ganciclovir is then injected into suitable brain regions of the transgenic rats to specifically eliminate or deplete acetylcholine, serotonin and / or noradrenaline neurons associated with a neo- or paleocortex expressing a negative selectable tk marker, to provide a rat model of Alzheimer's disease.

Claims (26)

A tissue derived from a transgenic eukaryotic organism, having cells comprising heterologous DNA, comprising a transgene encoding a negative selectable marker, wherein the tissue is substantially normal, except for a selectable phenotype derived from the transgene, for use in transplantation therapy, e.g. disorders. A tissue derived from a transgenic eukaryotic organism having cells comprising heterologous DNA comprising a transgene encoding a positive selectable marker wherein the tissue is substantially normal, except for a selectable phenotype derived from the transgene, for use in therapy. A tissue derived from a transgenic eukaryotic organism having cells comprising heterologous DNA comprising a transgene encoding a negative selectable marker and a transgene encoding a positive selectable marker for use in therapy. Tissue according to any one of claims 1 to 3, which is derived from a transgenic animal or plant, for example a vertebrate (e.g. a mammal, for example a rat, a rabbit, a pig or a mouse). A tissue according to any one of the preceding claims having a genotype that is substantially wild type, except for the presence of heterologous DNA and / or wherein the portion of the heterologous DNA that is expressed in cells consists of traWSJen encoding a positive selectable marker (and optionally trans). (encoding a negative selectable altered sheet marker), wherein each transgene is operably linked to a transcriptional element or elements. Tissue according to any one of the preceding claims, wherein at least one of the selectable markers is operably linked to a controllable expression element or element, for example a tissue or cell-specific expression element or elements. The tissue of claim 6, wherein each selectable marker is differently regulated, each marker being bound, for example, to a tissue or cell-specific expression element or elements. The tissue of any preceding claim, wherein the at least one selectable marker is constitutively expressed. The tissue of any preceding claim, wherein the heterologous DNA further comprises a reporter transgene, for example β-galactosidase or luciferase. The tissue of claim 9, wherein the reporter transgene is operably linked to an expression element or elements that are subject to cell or tissue-specific regulation. A tissue according to any preceding claim, wherein: (a) the positive selectable marker is selected from neomycin phosphotransferase, hygromycin phosphotransferase, xantinguanine phosphoribosyl transferase, herpes simples thymidine kinase type 1, adenine phosphoribosyltransferase and hypoxantine phosphoribosyltransferase, and / or the negative selectable marker genes selected from adrenaline selectin phosphoribosyltransferase, hygromycin phosphotransferase and hypoxanthin altered phosphoribosyltransferase leaf, and / or (b) the expression element is selected from: (I) promoters and / or enhancers. which are specifically active in: (i) dopaminergic, serotoninergic, GABAergic, cholinergic or peptidergic neurons and their subpopulations; (ii) oligodendrocytes, astrocytes and their subpopulations; (iii) endocrine glands, lungs, muscles, gonads, intestines, skeletal tissue or parts or portions thereof; (iv) epithelial cells, fibroblasts, fat cells, mast cells, mesenchymal or parenchymal cells; (v) individual stages of embryogenesis; (vi) blood system components (eg, T-lymphocytes, B-lymphocytes, and macrophages); or (II) promoters and / or enhancers that drive the transcription of genes for: (i) neurotransmitter specific receptors; (ii) ion channels; (iii) receptors involved in ion channel opening; and (iv) cytokines, growth factors and hormones. 12. A method for culturing cells and / or tissues in vitro, comprising the steps of: (a) providing a transgenic animal or plant having cells containing genetic material comprising a selectable marker that confers a selectable phenotype on cells, for example, a transgenic animal or plant containing tissue as defined in any one of claims 1 to 11; (b) generating a primary culture from the cells and / or tissues of the transgenic animal or plant of step (a); and (c) selectively cultivating the primary culture based on the selectable phenotype conferred by the genetic material altered leaf contained in the cells of the transgenic animal or plant. wherein the at least one selectable marker is operably linked to a tissue or cell-specific expression element or elements such that in step (c) a particular cell / tissue type is selectively cultured based on the tissue or cell-specific expression of said at least one selectable marker. 13. The method of claim 12, wherein step (c) produces a homogeneous population of a particular class of cells in the primary culture. A method of culturing cells and / or tissues in vitro, comprising the steps of: (a) providing a transgenic animal or plant having cells comprising genetic material comprising a positive selectable marker which confers a cell with a positive selectable phenotype, for example, a transgenic animal or plant, comprising tissue as defined in any one of claims 1 to 11; (b) generating a primary culture from the cells and / or tissues of the transgenic animal or plant of step (a); and (c) selectively cultivating the primary culture based on the selectable phenotype conferred by the genetic material contained in the cells of the transgenic animal or plant. wherein step (c) reduces or eliminates microbial (e.g. yeast or fungal) contamination of tissue culture. Tissues or cells cultured by the method of any one of the amended worksheets of claims 12 to 14, wherein the tissues or cells are intended for use, eg, as tissue transplants, as a test subject in biochemical assays, or as a source of the desired proteins. The tissues or cells of claim 15 for use in therapy. 17. A method of making monoclonal antibodies specific for an antigen comprising the steps of: (a) providing a transgenic animal (eg, a transgenic animal according to any one of claims 1 to 11), having lymphocytes that comprise genetic material that confers a selectable phenotype thereon; (b) immunizing the transgenic organism with an antigen; (c) collecting lymphocytes from the transgenic animal; (d) fusing the lymphocytes of step (c) with immortal cells (e.g., tumor cells, e.g., myeloma cells) to produce hybridomas; and (e) selectively culturing hybridomas based on a selectable phenotype conferred by genetic material contained in lymphocytes. The monoclonal antibody produced by the method of claim 17 for use in, eg, therapy. 19. A method for the selective elemination or depletion of certain tissues or cell types in an organism, comprising the steps of: the altered sheet (a) providing a transgenic organism comprising tissues as defined in any one of claims 1 to 11 having a negative selectable marker operably linked to an expression element (eg, a promoter) specific for the tissue or cell type to be specifically depleted; (b) administering a selective agent to the organism to eliminate or deplete tissue or cell type by expressing a negative selectable marker. The method of claim 19 for modeling the loss or atrophy of cells / tissues (eg, immunodegenerative or neurodegenerative diseases or disorders), characterized in that the tissue or cell type to be eliminated or depleted is that tissue or cell type that is a subject with a disease or disorder associated with elimination or atrophy. A transgenic organism comprising tissues as defined in any one of claims 1 to 11 for use in a method according to claim 19 or claim 20. An organism (eg, a vertebrate, eg, mammal) in which a particular cell / tissue type is specifically eliminated or depleted that is produced by the method of claim 19 or claim 20. The organism of claim 22, which is a model of a disease / disorder associated with atrophy or cell / tissue loss, eg, a model of immunodegenerative or neurodegenerative diseases / disorders. 24. A method of screening compounds for pharmacological activity against diseases or disorders involving atrophy or cell loss (e.g., neuronal loss and / or atrophy), a modified sheet comprising the steps of: (a) providing a disease test model according to the method of claim 20; (b) administering the compound to be tested to the test model of step (a); (c) examining the test compound for its effect on the test model. The method of claim 20 or claim 24, wherein the disease is: (a) Parkinson's disease and the tissue or cell type to be eliminated or depleted includes dopaminergic neurons in the substantia nigra; (b) Huntington's chorea tissue or cell type to be eliminated or depleted includes nerve cells of the striatum; (c) Alzheimer's disease and the tissue or cell type to be eliminated or depleted includes acetylcholine, serotonin and / or noradrenaline neurons associated with neo- and paleocortex; (d) multiple sclerosis and the tissue or cell type to be eliminated or depleted comprises brain oligodendrocytes; (e) the immune disease and the tissue or cell type to be eliminated or depleted comprises CD3, CD4 and / or CD8 cells; and the altered sheet (f) of AIDS and the tissue or cell type to be eliminated or depleted comprises CD4 cells. A method (eg, an in vitro method) of determining the effect of a first class cell deficiency (eg, brain cells) on second class cell characteristics in an organism, comprising the steps of: (a) providing a transgenic organism comprising tissues as defined in any one of claims 1 to 11 having a first negative selectable marker operably linked to an expression class specific for a first cell class and either: (i) a positive selectable marker operably linked to an expression element specific for a second cell class or (ii) a second negative selectable marker bound to an expression element that directs the expression of the negative selectable marker in all cells in the organism except the second cell class; (b) administering a selective agent to the organism to induce a first class cell deficiency by expressing a negative selectable marker; (c) harvesting the cells from the organism; (d) selectively cultivating second class cells from the cells harvested in step (c) based on: (i) their expression of a positive selectable marker, or (ii) the absence of expression of a negative selectable marker.