WO1998042824A2 - Methods for selecting cells and their uses - Google Patents

Abstract

Grafts, cells and tissues for use in transplantation, transgenic animals, methods of cell selection and various uses of such material. Use of induced apoptosis as a selectable negative marker for specific cell and tissue ablation avoiding local inflammatory response and 'bystander effect' (e.g. in engrafted tissues and/or cells). Models for tissue-specific degenerative diseases and disorders and screening methods for compounds active against those diseases related to a cell/tissue specific depletion of static or expanding cell lines.

Description

METHODS FOR SELECTING CELLS AND THEIR USES

The present invention relates to grafts cells and tissues for use in transplantatio to transgenic animals methods of cell selection and to various uses of such material In particular, the invention relates to cellular material derived from various organisms including humans, non- humans and transgenic organisms containing negative selectable marker can be induced to activate programmed cell death or apoptosis e g for use in transplantation therapy It is known in the art that cells can be routinely engineered or induced to express gene(s) which confer any of a wide variety of selectable phenotypes thereon Such genes are known as selectable markers They are normally introduced into cells as part of a recombinant expression vector The selectable phenotype conferred by a selectable marker may be classed as either positive or negative

A positive selectable phenotype is one which permits survival under particular conditions which would kill (or at least prevent or impair the growth of) cells which do not exhibit the positive selectable phenotype A negative selectable phenotype is one which results in the destruction (or the prevention or impairment oτ growth) of the cell under particular conditions which are relatively innocuous to cells which do not exhibit the negative selectable phenotype A negative selectable phenotype may be one which arises from the cell's own programmed cell death mechanisms eg the process of apoptosis

A wide variety of selectable markers are available Genes that are commonly applied as positive selectable markers include the bacterial neomycin phosphotransferase (neo, Colbere- Garapin et al (1981) 150 1), hygromycin phosphotransferase (hph, Santerre et al (1984) Gene 30 147) and xanthine-guanme phosphoπbosyl transferase (gpt Mulligan and Berg (1981 ) P N A S 78 2072)

Also used as positive selectable markers are the Herpes simplex virus type 1 thymidine kinase (HSV-1 TK, Wigler et al (1977) Cell 11 223), adenme phosphoπbosyl-transferase (APRT Wigler et al (1979) P N A S 76 1373) and hypoxanthine phosphoπbosyltransferase (HPRT Jolly et al (1983) P N A S 80 477) These latter markers must be used in cells having a particular mutant genotype (viz one which leads to a deficiency in the gene product on which the selection is based)

Some of the aforementioned genes also confer negative as well as positive selectable phenotypes They include the HSV-1 TK, APRT, HPRT and gpt genes These genes encode enzymes which can catalyse the conversion of certain nucleoside or puπne analogues to cytotoxic intermediates For example, the nucleoside analogue ganciclovir (GCV) is a good substrate for the HSV-1 thymidine kinase but a poor substrate for the natural thymidine kinase found in mammalian cells Consequently, GCV can be used for efficient negative selection against mammalian cells expressing the HSV- TK gene (St Clair et al (1987) Antimicrob 844) Xanthmeguanine phosphoπbosyl transferase can be used for e selection when expressed in wild type cells (Besnard et al (1987)

Selectable markers are usually used in both prokaryotic and eukaryotic genetic engineering to permit the recovery from a mixed population of cells which have unαergone a relatively rare genetic change For example, they can be used in physical association with another gene which encodes a product of interest (for example, a therapeutic protein) to select cells which have taken up that other gene along with the selectable marker For example, the neo gene has been used to monitor genetically modified cells taken from patient samples after gene therapy has taken place

It has also been proposed to use negative selectable markers as a safety device in gene therapy Many gene therapies involve the removal of somatic cells from the patient, the introduction therein of a therapeutic gene (the expression of which repairs a biochemical lesion), followed by reintroduction of the cells back into the patient Since the reintroduced genetically modified cells may ultimately prove deleterious to the health of the patient (for example, if they prove to be immunologically incompatible or become malignant), a negative selectable marker may be introduced along with the therapeutic gene to permit (if necessary) subsequent selective elimination of the genetically modified cells

A problem with the currently available negative selectable markers is the so-called bystander effect For example a cell which has been genetically engineered to express the viral thymidine kinase will convert GCV into a cytotoxic metabolite Similarly, cells expressing cytosine deamiπase will convert fluorocytosine (FC) to a cytotoxic intermediate also The intracellular build-up of these cytotoxic metabolites leads to the death of the engineered cell But, in addition death of non-engineered cells close to the engineered cell can also occur This is because cytotoxic ιntermedιate(s) leak from the dying engineereα cell into the local milieu, to be accumulated by the neighbouring non-engineered cells resulting in some cases in their own death

A second problem associated with the use of many of the currently availaDle negative selectable markers is their inefficient killing non-dividing cells under certain circumstances. It is well known that the cytotoxic effect of the intermediate generated by viral thymidine kinase from (e g GCV) is mediated by interfering with the cell's DNA synthetic pathway If the HSV-1 thymidine kinase-engineered cell is not undergoing DNA synthesis then GCV and its cytotoxic intermediate do not so effectively elicit cell death Similarly, only those cytosine αeaminase expressing cells which are also rep cative are most easily killed by FC aαministration Finally, the dosing regimen of known negative selectable agents such as GCV or FC in vivo needs to be of at least 10 days duration, and probably longer, in order to ensure complete eradication of all cells In a clinical situation, where total compliance with this regimen might need to be ensured and therefore closely monitored, such a duration of treatment is often undesirable

The invention mitigates these problems by providing inter alia methods for cell and gene therapy which permit the removal (if required) of engineered cells without a bystander effect, without the requirement that such engineered cells should be rephcative and with the possibility of a much reduced duration of administration of drugs to elicit the cytotoxic effect The method is particularly advantageous for neural cell transplantation and gene therapy in the central and peπpheral nervous systems (e g in the treatment of Parkinson's disease, Huntington s chorea Alzheimer's disease, amyotrophic lateral sclerosis, ischaemia-induced and trauma -induced cell loss), where a pruning or titration, or even a complete elimination of the grafted cells may become necessary, but without compromising residual function amongst the host cells Thus such a selection method limits bystander effects duπng the removal of the transDlaπted cells from the nervous system, and thereby attenuates damage to otherwise normal host tissue In addition, when the grafted or genetically engineered cells are non- dividing cells then the negative selection of the invention (which does not rely wholly or partly on cell αivision for its effects) is particularly advantageous Finally a more accurate pruning of cells numDers might be achievable if the drug required to elicit the cell death needs to be given over a short duration

The negative selectable markers of the invention exploit the natural mechanisms by which cells are decleted in vivo, for example during normal development These mechanisms are collectively known as programmed cell death, and are thought to underlie the process of apoptosis Apoptosis is a fundamental mechanism for regulating cell numbers at all stages of life in multicellular organisms. During embryogenesis, for instance, vaπous cells are selected for apoptosis, this leads to the manifestation of biological form, via either phylogenetic or morphogenetic shaping Programmed cell death also occurs in the normal adult For example the vertebrate haematopoietic system undergoes huge cell losses, with billions of neutropnils dying this way each day Apoptosis also plays a major role in tissue repair and regeneration when a cell is damaged beyond repair by e g ultra-violet irradiation, cell death is triggered using the cell's own suicide programme It is thought that this altruistic act helps to maintain the organism as a whole by guarding against further replication of damaged cells which may become cancerous, thereby leading to the death of the whole organism The control of aooptosis is currently a major component of worldwide research in cancer, since it is now be eveα that some cancers may result, not from an enhanced replication of tumour cells, but from a reαuced ability of such cells to undergo programmed cell death Another advantage of using a cell's natural mechanism of programmed cell death is that macrophages and microglia react to apoptotic cells in a highly specific fashion They engulf and digest apoptotic cells, but do not secrete inflammation-inducing signals, in contrast to their secretory profile when phagocytosing necrotic cells. Cells undergoing apoptosis rather than toxic cell death and necrosis, therefore, are not associated with a localized inflammatory response - a second unwanted contributor to the bystander effect of currently used negative selection markers

To activate the cell disposal mechanism, apoptotic cells change their surface chemistry so that macroDhages/microglia can recognize them The alteration may be in the surface lectins or in integnns and may vary depending on the cell type, but the final death is still via apoptosis

The invention rests at least in part on the recognition that the natural cellular mechanism of apoptosis can be used as a safety and/or titration switch to eliminate genetically engineered cells and in particular, cells transplanted to the central nervous system Using such a mechanism, it is possible to induce the apoptosis and subsequent elimination of grafted cells (e g in tne brain) This method is used advantageously, because such grafted cells may not necessarily be rephcative, (an at least partial requirement for ablation by negative selectable markers such as HSV-1 thymidine kinase or cytosine deaminase the grafted cells can be removed without the bystander effect of HSV-1 thymidine kinase due, for example to leaching of cytotoxic intermediates from the dying cells, and the localized inflammatory response they provoke Such a bystander effect could be particularly detπmental in the central nervous system

Another advantage is that apoptosis may be induced by a relatively short duration of treatment compared to the duration of treatment required to produce πon-apoptotic cell death Experiments indicate that e g up-regulation of biologically active p53 may induce apoptotic cell death within hours, by contrast, the cytosine deaminase or HSV-1 thymidine kinase negative selection mechanisms currently used require many days of treatment

Any negative selectable marker which can trigger programmed cell death may be used in the present invention For example, several factors are currently thought to be involved in the signalling and mediation of apoptosis In particular, apoptosis can result from increased expression of p53, stimulation of the cell surface Fas receptor, or activation of the interleukin- 1 b converting enzyme family of cysteine proteases In addition, c-myc, retinoblastoma, and the bax-bcl2 and related genes are involved in apoptosis These are the subject of reviews (Lin et al (1995) Cane Surv 25 173, Timmers & Kremer (1995) Biomed Rev 4 103, Hale et al (1996) Eur J Biochem 236 1 , Fraser et al (1996) Curr Op Neurobiol 6 71 , Shimamura & Fisher (1996) Clin Can Res 2 435, Vaux & Strasser (1996) P N A S 932239, Spence et al (1996) Exp Op Therap Pat 6 345, Gilardini et al (1996) Chn Immunotherap 5 413) which are incorporated as support In particular, we and others (Slack et al (1996) J Cell Biol 135 1085) have found overexpression of the tumor suppressor gene p53 to be of particular advantage in inducing the apoptosis of neural cells Any of the foregoing factors may be useα as the basis of the negative selectable markers according to the invention

Transgenic organisms bearing a selectable marker as a transgene are known in the art and are described, for example, in a pending patent application (PCT GB95 02586, WO 96/14401) which is incorDorated by reference herein These transgenic organisms may provide tissue and cells for transplantation, as well as having other uses Furthermore, a number of vectors bearing positive or negative selectable markers have been made and are readily available to those skilled in the art (for review see Miller (1992), Nature 357 455) Others may be readily assembled using standard gene cloning techniques

Another important problem arises from the need for cultures of a single tissue or cell type. Growth in vitro from single cells may be difficult (often requiring the use of feeder cells and/or mixtures of growth factors and other supplements) and homogeneous in vitro populations cannot therefore be easily obtained Homogeneous populations which also comprise a negative selection marker where programmed cell death can be induced are particularly difficult to produce

There is therefore a need for a convenient source of selectable cells/tissues of all types for primary culture or other purposes such as tissue transplantation and cell/gene therapy

It has now been found that transgenic organisms bearing a negative selectable marker which induces programmed cell death have previously unrecognized utility in cell culture techniques, and tissue transplantation and ceil/gene therapy

The present invention provides for, but is not limited to, transgenic organisms which inter alia constitute a very convenient source of material for the isolation, identification, culture and analysis of cells from any tissue of the organism's body Tissue dissected from the transgenic organisms of the invention can be particularly easily grown (even as homogeneous populations of a particular cell/tissue type) in vitro and used in a wide variety of applications, including pnarmaceutical assays, tissue transplantation, cell/gene the-apy general factor synthesis drug αelivery and protein production

For reasons of clarity transgene means the genetic structure that is transferred to the cell Although the term transgenic organisms has occasionally been applied to any organism which contains foreign DNA, the term "transgenic organism" is used herein in its more usual sense to denote eukaryotic organisms (and in particular, animals or plants, and especially vertebrates e g mammals) and their progeny which contain heterologous chromosomal DNA in the germ line The heterologous chromosomal DNA comprises a coding sequence which is hereinafter referred to as a "transgene" Thus, every (or at least most) of the cells of a transgenic organism - both somatic and germ - may contain one or more copies of the transgene(s)

Transgenic organisms can be produced by many different methods The methods are well documented in the prior art and their practice forms part of the technical repertoire of those skilled in the art Methodological approaches commonly used are descπbed for example in First and Haseltine (Eds ) Transgenic Animals (1991 ) Butterworth-Heineman MA USA

According to one known method, the transgene is inserted into embryonic stem cells which are then injected into fertilized zygotes at a stage when only a small number of cells are present The engineered embryonic stem cells become incorporated into the zygote, and cells derived therefrom go on to differentiate into many or all of the different cell types of the animal's body Sucn cells may also include those contributing to the germ ne, and the progeny of such (chimaeπc) animals may therefore be fully transgenic

Other methods involve the introduction of the transgene into the pronucleus or into the fertilized or unfertilized ovum, but the invention is not limited to the metnod of making the transgenic organism According to one asDect of the present invention, therefore, there is orovided a transgenic eukaryotic organism having cells containing heterologous DNA comDπsing a transgene encoding a negative selectable marker as herein defined But for the selectable phenotypes arising from the transgenes, the organism may be essentially normal (i e not mutant for any significant character or trait with respect to the wild type and/or in that the cells exhibit normal tissue differentiation and development), the transgenes not being located for example, such that they insertionally inactivate a gene

According to a second aspect of the present invention there are provided eukaryotic cells (e g as in pending patent applications PCT/GB95/02592 and PCT/GB96/00671) containing heterologous DNA comprising a transgene encoding a negative selectable marker as herein defined But for the selectable phenotypes arising from the transgenes, the cells may be essentially normal (i e not mutant for any significant character or trait with respect to the wild type and/or exhibiting normal tissue differentiation and development) According to a third aspect of the present invention, therefore, there are provided eukaryotic immortalized cells (e g as in pending patent applications PCT/GB95/02591 and PCT/GB95/02497) containing heterologous DNA comprising a transgene encoding a negative selectable marker as defined herein But for the selectable phenotypes aπsing from the transgenes the cells may be essentially normal (i e not mutant for any significant character or trait with respect to their phenotype prior to incorporation of the selectable marker and/or exhibiting normal tissue differentiation and development)

According to a fourth aspect of the present invention, therefore there is provided eukaryotic cells showing any advantageous phenotype and containing heterologous DNA comprising a transgene encoding a negative selectable marker as defined herein But for the selectable phenotypes arising from the transgenes, the cells may be essentially as before transgenesis of the selection marker (i e unchanged in any significant character or trait with respect to the original advantageous phenotype and/or exhibiting normal tissue differentiation and development)

As stated above the term "essentially normal" as used herein may indicate that the cell or organism is not mutant for any significant character or trait with respect to the wild type or the phenotype prior to the insertion of the selectable marker, and/or exhibits normal tissue differentiation and development The term ' essentially normal" therefore includes transgenic organisms or cells constructed so as to provide essentially normal cells for transplantation Since cells considered to be less immunogenic to a host, should tney be implanted, can still exhibit normal tissue differentiation and development, they too would be essentially normal, as herein defined Tne organism may also be essentially normal in tne sense that the transgenes are resident in a silent (i e non-expressed region of the genome and/or in a region of the genome wnere transgenes do not significantly perturb the replication, segregation, organization or packing of the chromosome or its interaction with cellular components such as DNA binding prctε'ns (including histones and regulatory elements)

The provision of transgenes encoding both a negative selectable marker as herein defined and a positive selectable marker provides great flexibility during subsequent manipulation of any eukaryotic cells of the invention in vitro Moreover, where the invention is used to generate tissue transplants, cells of a particular type may be isolated from e g a transgenic animal of tne invention by positive selection The cells so isolated then may be transplanted into a non-transgenic animal to determine whether the transplant has any therapeutic effect The transplant may be ablated by the negative selection of the invention to provide a control to determine wnether the transplant was having a direct therapeutic effect

It will beclear to those skilled in tthe art that a cell suicide mechanism could be applied to all forms of cell/tissue grafting where it is advantageous to deplete or destroy the graft, or cells from the graft Tnis would be of particular advantage when the cells to be depleted/destroyed undergo little or no replication as would be expected for so-called static or expanding cell populations In terms of cell kinetics there are three populations of cells Renewal cell populations such as those of the gut or skin, whereby progenitor and stem cells are continually dividing to produce differentiated cells which perform their required functions, and which, after a period of functioning, are lost by eg exfoliation Expanding cell populations do not normally divide However under certain conditions, such as hormone stimulation or wounding, the cells will divide For example, the thyroid gland will increase in cell number in response to thyroid stimulating hormone Also, liver ceils will replicate in response to damage, and indeed will replace their entire cell population if eg 75% of the liver is removed However, in static cell populations such as neurones or cardiac muscle, cells do not divide If a graft were to comprise static or expanding cells, then, it will be seen that the invention has a particular advantage

The invention would allow cells from the graft, or the graft as a wnole, to be depleted or destroyed readily in the absence of cell replication, by virtue of programmed cell death not requiπng replication for its initiation

The invention would be of value when any cell type or cell engineered to express a phenotype having any advantage is used, eg cells or tissue or organs for grafting which are functionally normal but have been humanized le to make them immunologicaliy more tolerable to the human host receiving the graft The invention would also be of value when cells engineered to express proteins/factors more relevant to the human, or to the alleviation of a human medical condition than the unmodified tissue source, are used The cells/organs/tissue comprising the invention would be for use as eg ailogeneic, homogeneic and xenogeneic grafts The invention would have particular advantage in neural grafting, cardiac grafting, hepatic grafting, vascular grafting, thyroid grafting and pancreatic grafting, whether xenogeneic grafting of humanized tissue/cells, or ailogeneic grafting or homogeneic grafting or other

In another aspect the invention provides cells or a transgenic eukaryotic organism having cells containing heterologous DNA comprising a transgene encoding a negative selectable marker of the invention as herein defined and a positive selectable marker, the organism or cells might be essentially normal as herein defined but for the selectable phenotypes arising from the transgene(s)

The cells of the invention are preferably animal cells, for example a vertebrate (e g a mammal, for example a rat, rabbit, pig or mouse) The transgenic organism or cells preferably may have a genotype wnich is essentially normal as herein defined but for the presence of the heterologous DNA. Alternatively, the selectable cells of the invention may be derived from human or any non-human source and may not be essentially normal but be hybrids and/or express any phenotype In addition, that portion of the heterologous DNA which is expressed in the cells may consist of a transgene encoding a positive selectable marker and a transgene encoding a negative selectable marker as herein defined, each transgene being operaDly linked to an expression element or elements The absence of expression of any other transgenically derived genetic sequences makes this a preferred transgenic organism suitable for a wide range of experimental research and cell/tissue/organ transplantation therapy requiring an effectively wild type genetic background

A further aspect of the present invention is that the heterologous DNA which constitutes the negative selectable marker as defined herein may additionally include a multiplicity of alternative positive and/or negative selectable markers, including two or more negative selectable markers as defined herein

At least one of the selectable markers may be operably linked to a regulatable expression element or elements, for example a tissue- or cell-specific expression element or elements In such circumstances, each selectable marker is advantageously differentially regulated, each marker for example being linked to a different tissue- or cell-specific expression element or elements This permits the expression of the selectable marker to be limited to a selected class of cells or tissue, or to be limited by temporal expression e g during a specific stage of embryogenesis, so providing e g for the selective culture in vitro of the selected class of cells or tissue from a mixed primary cell culture The present invention does not rely on the use of cells with transgenes or transgenic organisms produced by any one method any transgenic procedure may be used in the practice of the invention Moreover, in most circumstances, the precise nature of the selectable markers for use in the present invention is unimportant in general, any selectable marker gene may be used so long as it additionally confers a negative selectable phenotype as herein defined on the cell

For example the positive selectable marker may be selected from neomycin phosphotransferase, hygromyαn phosphotransferase, xanthineguanine phosphoπbosyl transferase, the Herpes simplex virus type 1 thymidine kinase, adeπine phosphoπbosyltransferase and hypoxanthine phosphoπbosyltransferase

The negative selectable marker may, for example, be selected from any of the factors known to induce cells expressing it to undergo programmed cell death (apoptosis) Those factors might include p53, interleukin 1 b converting enzyme (ICE) cysteine proteases, retinoblastoma (Rb) or mutants thereof c-myc, bax, Fas, Fos, poly(ADP)πbose polymerase (PARP), cpp32 or YAMA or any other factor(s) or combinations thereof which induce cell death by apoptosis rather than cytotoxicity

The selectable markers are conveniently derived (e g by subcloning using restriction endonucleases) from any of a large number of known vectors, examples of wnich are described m e g Molecular Cloning A laboratory Manual Second Edition Edited by Sambrook J, Fπtsch and Maniatis T 1989 Cold Spring Harbour Laboratory Press) or from ATCC, or GenBank and/or EMBL databases, where vectors comprising the required selectable markers can be obtained or information required to generate or clone the required sequences can be retrieved, and/or from the literature

The expression elements for use in the invention may take any form so long as they can (under at least some circumstances) be made to direct and/or control the expression of the genes to which they are operably coupled Expression elements for use in the invention may comprise transcπptioπal and/or translational elements, and include promoters, πbosome binding sites enhancers and regulatory sites including activator and repressor (operator) sites Preferred expression elements comprise promoters selected from a wide range available for use examples of which are shown in Table 1 This Taole, wnich is non- exhaustive, also indicates the use to which each promoter may be put in the methods of the invention described infra

By way of example only, the expression elements for use in the invention may be selected from promoters ano/or enhancers which are specifically active in (i) dopaminergic, serotoninergic, GABAergic, cholmergic or peptidergic neurones and sub-populations thereof, (n) ohgodendrocytes, astrocytes and sub-populations thereof, (in) the endocπne glands, lungs, muscles, gonads, intestines, skeletal tissue or part or parts thereof, (iv) epithelial, fibroblast, fat, mast, mesenchymal or parenchymal cells, (v) particular stages of embryogenesis, and (vi) components of the blood system (e g. T-lymphocytes, B-lymphocytes and macrophages) Alternatively they may be selected from promoters and/or enhancers which direct the transcription of genes for (i) neurotransmitter-specific receptors, (u) ion channels, (ni) receptors involved in ion channel gating and (iv) cytokmes, growth factors and hormones Additionally, the expression elements for use in the invention may be inducible promoters which could take many forms e g as in Jones et al (1991 ) Nucl Acids Res 19 6547, Woodroofe et al (1992) DNA & Cell Biol 11 587, Muller et al (1992) Gene 121 263, Simson et al (1994) Lab Invest 71 680, Blessing et al (1995) Terat Care Mutagen 15 11 , Shockettt al (1995; P N A S 92 6522 Hoff et al (1995) J Am Soc Nephrol 6 793, Maxwell et al (1996) Gene Therap 3 28, Walther & Stein (1996) J Virol 70 6054 Ho et al (1996) Mol Brain Res 41 200 Delort & Capecchi (1996) Hum Gene Therap 7 809 or may be the Tet- offTM or Tet-onTM system currently marketed by Clontech (see Figure 1) which has been shown to be effective in both cells (Gossen et al (1995) Science 265 1766) and transgenic organisms (Kutner et al (1996) P N A S 193 10933) and additionally effective in brain (Mayford et al (1996) Science 274 1675)

At least one of the selectable markers may advantageously be coπstitutively expressed This ensures uniform expression of the selectable marker in every transgenic cell of the transgenic organism under all conditions, which is particularly useful where the transgenic organism is for general use as a source organism for cell/tissue culture

Constitutive expression may be achieved for example via the use of a promoter which directs the expression of a "house-keeping" gene A "house-keeping" gene is one which is expressed in all cell types Their translated products are required as part of general cell metabolism or cell structure and consequently, they are not specifically expressed in a particular cell or tissue type House-keeping gene promoters, therefore, need to be active in a broad range of (and sometimes in all) cell types in order to ensure constitutive gene expression When constitute promoters are used in the invention, then alternative regulation of expression of genes may be necessary The constitutive expression could be regulated by temperature- sensitive mutants of the promoter/enhancer elements so that expression is only allowed for cells incubated within a defined temperature range Alternatively, expression may be controlled by the requirement for factors in the medium or milieu of the cells or in the diet of the organism by the adminsitration of factors to the organism, the absence of which would not allow active expression of genes involved in programmed cell death Alternatively, the presence of specific factors may be required to inhibit promoter/ennancer directed expression of genes involved in programmed cell death Control may also be achieved by regulating the biologically active form of the factor(s) required to induce apoptosis The invention is, therefore not limited by the method used to modulate active epxression of factors required to induce programmed cell death

An example of a coπstitutively-expressed promoter useful in the present invention is that for the histocompatabi ty complex H-2Kb class 1 promoter (Weiss et al (1983) Nature, 301 671; Baldwin and Sharp (1987), Mol Cell Biol 7 305, Kimura et al (1986), Cell 44 261 ) which has been shown to express downstream coding sequences in cells generally when used as a promoter in a transgene (Jat et al (1991 ), P N A S 88 5096) Another example is the viral SV40 early promoter

The promoters for use in the present invention are not restricted to those derived from mammalian cells but may also include avian- and fish-derived promoters Additionally, virally deπved promoters, some of which have biological activity in a broad range of mammalian, fish and avian cells as well as other eukaryotes, could also be used in performing the invention Examples are the simian vιrus-40 derived early or late promoters, or the Long Terminal Repeats (LTR'S) of retroviruses which comprise promoter as well as enhancer elements and have the ability to promote expression of sequences under their influence in a broad range of eukaryote cells These promoters along with supporting sequences such as enhancer elements and other regulatory elements are well known to the man skilled in the art (see e g Molecular Cloning A laboratory Manual Second Edition Edited by Sambrook J, Fntsch and Maniatis T (1989) Cold Spring Harbour Laboratory Press) The cells or transgenic organιsm(s) of the invention may also contain heterologous DNA which further comprises a reporter transgene, for example 3-galactosιdase or luciferase The reporter transgene may be itself operably linked to an expression element or elements which are subject to cell- or tissue-specific regulation Such reporter transgenes facilitate subsequent analysis of cells/tissue cultured from e g the transgenic organism and in particular permit the response (to for example an induced deficit in a particular class of cells/tissue) of a particular expression element or class of expression elements to be monitored in vivo or in vitro In another aspect the invention provides a method of cultuπng cells and/or tissues in vitro, comprising the steps of (a) providing a cell or a transgenic organism having cells containing genetic material which confers a negative selectable phenotype, as nerein defined, thereon, (b) generating a pπmary culture of cells or a cell line or from cells and/or tissue of the transgenic organism of step (a), (c) transplanting said cells and (d) when required, selectively removing the said cells on the basis of the selectable phenotype conferred by the genetic material contained in the primary cells or the cell line or the cells of the transgenic organism

Preferably, the cell/tissue culture method of the invention is based on the use of primary cells or cell lines or a transgenic organism having a selectable marker which incorporates the upstream selectable marker operably linked to a tissue- or cell-specific expression element or elements, whereby in step (d) a particular cell/tissue type is selectively grown on the basis of the tissue- or cell-specific expression therein of said at least one selectable marker

This preferred method of the invention finds application for example in the selection of thyroid follicular cells from a primary (mixed cell) culture This method may provide a primary stromal cell population of the thyroid gland in the absence of the thyroid follicular cells and constitutes a unique cell culture system useful for the study of thyroid biology and in the development of new therapeutic drugs for the treatment of thyroid diseases The transgenic organisms of the present invention also find application in relation to diseases involving cell loss

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

In Parkinson's disease, this loss is principally of the dopammergic neurones of the substantia nigra region of the brain, although other cell types also decline

In Huntington's chorea, there is a more general loss of neurones, but in this case the deficits are restricted largely to the stπatum

In Alzheimer's disease, there is a decrement in acetylcho ne-, serotonin- and noradrena ne- containing neurones projecting to the neo- and palaeocortex Other neuroiog.cal diseases also stem from neural cell degeneration the demye nation occurring ιr multiple sclerosis, for instance, is due to the destruction of oligooendrocytes in the brain

The Human Immunodeficiency Virus (HIV) is known to enter cells that express the CD4 receptor aπo cell infection appears to lead ultimately to cell death The loss of CD4 cells causes a catastrophic block of the entire immune system and death of the infected person The molecular/cellular basis of HIV induced-disease is poorly understood This is due, at least in part to tne lack of model systems to study the pathogenesis of the disease, particularly in vivo

The use o SIV (simian immunodeficiency virus) infected primates has been considered as a paradigm put SIV monkeys do not acquire full-blown AIDS In many instances, they show no symptoms at all Alternative models that have been proposed include HIV-infected chimpanzees Apart from the potential ethical considerations, the manisfestation of AIDS-like symptoms in such a model may take several years, substantially hindering research and the development of effective therapies

Thus, animal models of the various diseases discussed above are essential as test subjects for potential orarmaceuticals and in basic clinical research The choice of these animal models is presently very limited because of the difficulties associated with selectively destroyinα soec.fic cell and/or tissue types

Thus, according to a further aspect of the present invention there is provided a method of selectively eliminating or depleting a particular tissue or cell type in an organism, comprising the steps of (sj providing a transgenic organism having a negative selectable marker as defined herein operably linked to an expression element (e g a promoter) specific for the tissue or cell type to be eliminated or depleted, and (b) administeπng or ceasing to administer a selective agent to the organism to eliminate or deplete that tissue or cell type on the basis of the expression therein of the negative selectable marker as defined nereiπ The selective agent is prefe^aole one which induces or activates the negative selectable marker to induce programmed ceil death

The selective agent may be administered by any route Where systemic administration is required oral oarenteral or intravenous routes may be used Where localized administration is required (for example where the tissue or cell-type to be eliminated is restπcted to a particular organ or to a particular region of the body) targeted injection, implantation (e g slow release cacsuies) or catheteπzatioπ may be used For example tissue in particular regions of the brain may be specifically targeted by intracerebral injection

The method of selectively eliminating or depleting a particular tissue or cell type of the invention may oe employed to provide in vivo models of diseases involving disease-related cell loss

Accordingly in a further aspect the present invention provides a metπod of modelling disease- related cell/tissue loss or atrophy comprising the steps of (a) providing a transgenic organism having a negative selectable marker as defined herein operably linked to an expression element (e g a promoter) specific for the tissue or cell type which is subject to disease-related elimination or atrophy and (b) administering or ceasing to administer a selective agent to the organism to eliminate or deplete the tissue or cell type on the basis of the expression therein of the negative selectable marker The invention also provides a method (e g an in vitro method) of determining the effect of a deficit in a first class of cells on the characteristics of a second class of cells in an organism, the metnod comprising the steps of (a) providing a transgenic organism having a first negative selectable marker as defined herein operably linked to an expression element specific for the first class of cells and either, (i) a positive selectable marker operably linked to an expression element specific for the second class of cells, or (n) a second negative selectable marker linked to an expression element which directs the expression of the negative selectable marker in all cells of the organism except the second class of cells, (b) administering a selective agent to the organism to induce a deficit in the first class of cells on the basis of the expression therein of the negative selectable marker (c) removing cells from the organism, and (d) selectively cultuπng cells of the second class from those cells removed in step (c) on the basis of, (i) the expression therein of the positive selectable marker, or (n) the lacκ of expression therein of the negative selectable marker

In another aspect the invention provides a method of screening compounds for pharmacological activity against a disease involving cell/tissue loss or atrophy, comprising the steps of (a) providing a test model of the disease via the steps of, (i) providing a transgenic organism having a negative selectable marker as defined herein operably linked to an expression element (e g a promoter) specific for the tissue or cell type which is subject to disease-related elimination or atrophy, and then (n) administering a selective agent to the organism to eliminate or deplete the tissue or cell type on the basis of the expression therein of the negative selectable marker to produce a test model, (b) administering the compound to be tested to the test model (c) screening the compound to be tested on the basis of its effect on the test model of step (a) The methods of the invention may be usefully applied to any disease which is associated with cell/tissue loss or atrophy In particular, the methods of the invention find particular utility in respect to (a) Parkinson's disease (the tissue or cell-type to be eliminated or depleted compnsing dopaminergic neurones in the substantia nigra), (b) Huntington's chorea (the tissue or cell-type to be eliminated or depleted comprising neural cells of the stnatum, (c) Alzheimer's disease (the tissue or cell-type to be eliminated or depleted comprising acetylcholine-, serotonin- and/or noradrenahne- neurones associated with the neo- and palaeocortex, (d) multiple sclerosis (the tissue or cell-type to be eliminated or depleted compnsing brain ohgodendrocytes), (e) immune disease and the cell-type to be eliminated or depleted comprises CD3, CD4 and/or CD8 cells and (f) AIDS and the cell-type to be eliminated or depleted comprises CD4 cells

In the case of AIDS models, the method of the invention could be used to specifically deplete or eliminate CD4 cells by linking a negative selectable marker as defined herein to a CD4 cell- specific promoter (e g the CD4 receptor promoter) This would permit the generation of an in vivo model of AIDS by regulating the proportion of cells expressing CD4 by negative selection

Furthermore, in the case where the transgenic animal model carries both a positive and negative selectable marker as defined herein, any residual CD4 expressing cells could later be isolated from the transgenic tissue of the animal model by positive selection in vitro for further study Also the cells/tissues/organs which may be of animal or human oπgin, of the invention may be usefully applied to human therapeutic areas such as Parkinson's disease, Huntington s chorea Alzheimer's disease, stroke injury, diabetes, kidney, heart and liver dysfunction

Examples of various promoters suitable for use in the methods of the invention described above are listed in Table 1 , along with the dιsease(s) in which each promoter may find application The invention also contemplates cell/tissue cultures derived from the transgenic organisms of the invention (or produced by the cell culturing methods of the invention), and also to various therapeutic uses of the invention The ability to trim or destroy cells/tissues/organs transplanted to e g humans by the method of the invention has a particular selective advantage

The invention will now be described in more detail by way of specific examples These examples are not intended to be taken as limiting in any way The examples are of proposed protocols which, without wishing to be bound by any theory, it is believed could be practised (with or without modification) by those skilled in the art The constructed sequences given below represent examples of numerous constructs that could be used to perform the invention The invention should not be construed as being limited to their use only

Materials Vectors pBabeneo plasmid vector Morgenstem & Land (1990) Nucl Acids Res 18 3587

(plasmid freely available) pCI plasmid vector Promega, 2800 Woods Hollow Rd, Madison, USA CD2 plasmid vector Blaese MR, NIH, Bethesda, USA (plasmid freely available)

Mullen et al (1992) P N A S 89 33, Austin & Huber (1993) Mol Pharmacol 43 380, Wallace et al (1994) Cancer Res 54 2719 TG-TKa plasmid vector Wallace H, Kings Buildings, University of Edinburgh, UK (plasmid freely available) Wallace et al (1991 ) Endocrinology 129 3217 pPBS plasmid Morgan (1992) Nucl Acids Res 20 1293

Plasmid comprising wild-type p53 etc Baccheti & Graham (1993) Int J Oncol 3 781 , Katayase et al

(1995) Cell Growth Diff 6 1207, Lin et al (1995) Cancer Surv 25 173, Timmers & Kremer (1995) Biomed Rev 4 103, Hale et al (1996) Eur J Biochem 236 1 , Fraser et al (1996) Curr Opin Neurobiol 6 71 , Shimamura & Fisher (1996) C n

Cane Res 2 435, Vaux & Strasser (1996) P N A S 93 2239, Spence et al (1996) Expert Opin Therap Pats 6 345, Gilardiπi et al (1996) Clin Immunotherap 5 413, Moore & Thanos (1996) Prog Neurobiol 48 441 98

-14-

Molecular Biology Reagents

Restriction endonucleases Promega, Madison, USA

DNA modifying enzymes, Promega, Madison, USA ligase, CIP, T4 polymerase etc

Agarose for electro- Sigma Chemical Co., St. Louis, USA phoresis

Polynucleotide kinase and New England Biolabs Ltd., and buffers 3397 American Drive, Unit 12,

Mississauga, Ontario, Canada

Construction of genes Thyroglobuliπ-thymidine kinase-internal πbosomal entry site-neomycin resistance (TG-TK-a- IRES-neor)

The neomycin resistance gene (neor) was obtained from the pBabe Neo plasmin (Morgenstem & Land (1990) Nucl. Acids Res. 18:3587) by digestion with Hind lll/Cla I and retrieval for the 1165 b.p. fragment containing neor gene by gel eleictrophoresis and the Promega Wizard PCR kit.

The pPBS plasmid (Morgan (1992) Nucl. Acids Res. 20:1293) compnsing the poliovirus- derived internal πbosomal entry site sequence was digested with Hind lll/Cla I. However, this could not be done simultaneously or in sequence, since the restriction sites were too close together. In order to overcome this problem, the plasmid was initially digested with Hind III and a 200 b p. fragment of DNA containing Hind III restriction sites at both the 5' and 3' ends was inserted in order to separate the sites. The pPBS plasmid couid then be digested first with Cla I and then with Hind III.

Terminal phosphate groups were removed from the Hind lll/Cla I cut pPBS vector using calf intestinal phosohatase (CIP). The vector was gel-purified using a 1 % agarose gel and a band containing the DNA was excised and electroeluted. The neomycin gene was then ligated into the pPBS plasmid overnight at 15° C and the ligation reaction transformed into freshly-made MC1061 competent cells.

Positive colonies were identified by digestion of prepared plasmids with Hind lll/Cla I. The neor gene and plasmid being detected electro-phoretically in plasmid preparations from positive colonies. Plasmids from the positive colonies were then digested with Hinc II and Sac I (both restriction enzymes leaving digested DNA with blunt ends). The resulting Sac l/Hinc II digestion containing the IRES-neor fragment was run on a 1 % elecuophoresis gel and the appropriate size band was excised and the DNA electroeluted and ethanol-precipitated. The TG-TKa plasmid (freely available from Genbank, NIH, USA accession No. J02224, Santelli et al. 1993) DNA was prepared using Promega Wizard mini preps and digested with Nar I. The ends of the plasmid were blunted using T4 Polymerase at 37° C for 1h followed by ^■ removal of the terminal phosphate groups using CIP. The CIP was inactivated by treatment of the DNA with phenol-chloroform followed by ethanol precipitation. The resulting plasmid was electrophoresed on a 1% agarose gel and the DNA was recovered and ligated with the insert ιn a 1 3 molar ratio of plasmid to insert

The ligation was incubated at 15° C overnight, and was then used to transform competent MC1061 cells Positive colonies were selected by digestion of prepared plasmids with BamH (the correct construct provided restriction fragments of size 3980, 1663 3102 and 1039 b p )

Linearization of the plasmid was achieved by digestion of prepared plasmids with Sal I restriction enzyme The construction is shown in Figure 2 Thyroglobulin-wild type p53-a-IRES-neor (Tg-p53-a-IRES-neor)

This plasmid was constructed as (ι)(a) above but wild type-p53 in plasmid form was obtained from Prof J Milner, University of York, UK and inserted in place of TK Cytomegalovirus-cytosine deamιnase-SV40 promoter-neomycin resist-ance (CMV-CD-SV40- neor, or CD2-neor) pCD2 plasmid (Mullen et al (1992) P N A S 89 33) was digested with EcoR I and EcoR V, and the digest was electrophoresed on a 1% agarose gel where the 2 5 kb fragment containing the cytosine deaminase gene, the SV40 promoter and the neomycin resistance gene was retrieved by electroelution followed by ethanol precipitation

To ensure terminal phosphate groups were present in the fragment it was treated with polynucleotide kinase

The pCI vector was digested with EcoR 1 and Sma I (a restriction enzyme leaving the DNA with blunt ends), and the terminal phosphate groups were removed using CIP and the enzyme was inactivated with phenol/chloroform followed by ethanol precipitation The band was then gel-purified and recovered by electroelution

The ligation was set up containing a 3 1 molar ratio of insert to vector and was earned out at 15° C overnight The ligation mixture was used to transform freshly-prepared MC1061 competent cells and positive colonies were selected by digestion of prepared plasmids with EcoR 1 and Hind III to provide restriction fragments of length 1868 b p and 5062 b p , respectively Linearization of the plasmid was achieved by digestion with Bgl I

The construction is shown in Figure 3

Cytomegalovirus-wild type p53-SV40 promoter-neomycin resistance (CMV-p53-SV40-neor)

The plasmid was constructed as in (n)(a) above, but with p53 inserted in place of CD

A number of other plasmid constructs can be prepared using the techniques and methods outlined in the construction of the plasmids of ι(a), ι(b), ιι(a) and ιι(b) above All methods required to construct the plasmids are well known to the artisan of applied molecular genetics and genetic engineering

The constructs, for example, using the Tet-OnTM system available from Clontech are as follows 1a CMV oromoter-rtetR-VP16 (this plasmid also confers neomycin resist-ance to the host cell)(see Clontech literature) 1 b TRE-Pmin CMV-wild type p53

Both plasmids (1a and 1 b) are incorporated into the same cell or to construct a transgenic organism The cell, or cells of the organism, will express wild type p53 in the presence of e.g. doxycychne

Chohne acetyl transferase (CAT) promoter-rTetR-VP16 and 1 b. are incorporated into the same cell or used to construct a transgenic organism, such that both 2 and 1 b are incorporated into cells in the transgenic organism Cells which normally express CAT will also express wild-type p53 in the presence of e.g. doxycychne

CAT promoter-neor-CMV promoter-rTetR-VP16 and 1 b are incorporated into the same cell or used to construct a transgenic organism, such that both 3 and 1 b are incorporated into cells in the transgenic organism. Cells will express neomycin resistance under the control of the tissue-specific CAT promoter, allowing for positive selection of CAT-expressing cells. Such cells will also express wild-type p53 in the presence of e g. doxycychne. This provides an example of tissue-specific positive selection with negative selection, but this should not be seen as limiting since, given an understanding of the invention, the man skilled in the art could link any specific positive selection marker to create an aspect of the present invention

The plasmids of 1b. where the wild-type p53 is replaced by ICE or Rb or mutants thereof, bas, Fas, Fos PARP, cpp32 or YAMA or any other fator(s) which induce programmed cell death, or combinations thereof by e.g. constructs with e.g. internal πbosomal entry site separation units or multiples of the section 3 constructs.

Plasmids comprising a cell suicide gene such as p53 operatively linked to an inducible promoter as exemplified in the following publications: Jones et al (1991 ) Nucl Acids Res. 19.6547; Woodroofe et al. (1992) DNA & Cell Biol.

11.587, Muller et al. (1992) Gene 121 :263; Simson et al. (1994) Lab. Invest. 71 680, Blessing et al. (1995) Terat. Care Mutagen. 15:11 ; Shockettt al. (1995) P N.A.S. 92^"6522; Hoff et al.

(1995) J Am Soc. Nephrol 6 793; Maxwell et al. (1996) Gene Therap. 3.28; Walther & Stein

(1996) J Virol 70 6054, Ho et al. (1996) Mol. Brain Res. 41.200, Delort & Capecchi (1996) Hum. Gene Therap 7.809 or may be the Tet-offTM or Tet-onTM system currently marketed by Clontech (see Figure 1) which has been shown to be effective in both cells (Gossen et al. (1995) Science 265.1766) and transgenic organisms (Kutner et al (1996) P N.A.S. 193 10933) and additionally effective in brain (Mayford et al. (1996) Science 274' 1675). Production of transoenic animals

Transgenic rats were produced by established methods (Hogan et al. (1986) Manipulating the Mouse Embryo - A Laboratory Manual, Cold Spring Harbor Lab., Cold Spπng Harbor, N.Y.). In brief, approximately 2 pi of the plasmid were microinjected at a concentration of 5 μg/ml into the pronucleus of outbred Sprague-Dawley embryos. Embryos were then implanted into pseudopregnant recipients, and after identification of transgenic animals, lines were isolated and established Lines were maintained as transgenic hemizygotes by mating hemizygous females with non-transgenic males. Positive/negative selection of cells from transgenic animals in vitro. Fibrobiast cells.

Fibroblast cultures derived from lung of adult CD2/neor, TGTK neor and control animals were produced and expanded by routine 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 from control rats, and replaced every three days with fresh medium When required, cells were subcultured (1 :3) to prevent them becoming confluent, again by basic culture methods (Freshney 1987). Cell counts were made manually in 20 fields chosen randomly and the values at each time point, after allowing for changes due to subcultuππg, were aggregated. As can be seen from Table 2, no fibroblast cells derived from control animals or the TG TK neor transgenic survived more than 10 days treatment with geneticin In the absence of added geneticin, no change in cell survival from either of the transgenic animals was observed.

The effects of 5-fluorocytosine (5FC) were also determined. 5-fluorocytosine at a concentration of 100 μg/ml had no effect on fibroblast cells derived from control animals or from the TGTK neor transgenic. In the cells derived from the CD2/neor transgenic animal, however. 94% of the orginally-plated cells died, or were non-functional (as determined by their failure to exclude trypan blue) after 10 days culture in the presence of 5FC (Table 2). By contrast, no significant difference in cell counts was found between cultures from control rats in the aosence and presence of 5FC, or between controls and cultures taken from CD1/neor rats in tne absence of added 5FC (Table 2). Thyroid cells

Thyroid cultures derived from the thyroid gland of adult CD2/neor, TG/TK/neor and control animals were produced by routine methods (Freshney, 1987). Twenty-four hours after plating, geneticin (400 μg/ml) was added to cultures originating from both types of transgenic and the control rats, and replaced every three days with fresh medium. When required, cells were subcultured (1 :2) to prevent their becoming confluent. Cell counts were made manually in 20 fields chosen randomly, and the values at each time point, after allowing for changes due to subcultuπng, were aggregated (Table 3). Ten days after the initial application of ganaticin, 10 μg/ml acycloguanosine (ACG, Sigma) was added to thyroid cells originating in the TG/TK neor transgenic. Ten days later, cell counts were again made of 20 fields chosen at random. Results are given in Table 3. To summarize, cells derived from both types of transgenic animal survived the geneticin treatment, whereas the control cells did not. Cells derived from the TG TK/neor transgenic did not survive ACG treatment, whereas the cells derived from the control animals did. The results were as expected in view of the specificand non-specific expression of the positive and negative selection markers, in the TGTK neor and CD2/neor transgenics, respectively. TG/TK/neor transgenic rat thyroid cells cultured in the absence of any aoced drug did not exhibit any differences intheir growth or survival compared to control thyroid cell cultures (Table 3). Ablation of thyroid follicle cells in vivo

Adult female rats (250g) were injected intra-peritoneally with 50 mg of ACG per day for a period of 5 days. Seven days after the final injection, serum levels of T3 and T4 were measured (Amersham, UK), and found to have fallen in transgenic animals from 0.76 @ 0.05 nM to less than 0.06 nM (T3) and from 58.2 @ 3.2 nM to less than 2.5 nM (T4) (N=6). Administration of saline to transgenic animals resulted in small but non-significant fall in T4 to 0 68 @ 0 07 nM (N=6) The thyroid glands of transgenic rats treated with ACG for 12 days had shrunk to 7% of the original weights Histochemical analysis of these thyroid glands revealed an almost complete loss of follicular cells, with only non-follicular, perhaps calcitonin- producing cells remaining Administration of lower amounts of ACG per day resulted in a partial loss of T3 and T4 In most other tissues from transgenic animals, HSV-thymidine kinase activity (Bπnster et al (1981) Cell 27 223, Jamieson et al (1974) J Gen Virol 24 481) was not expressed in detectable amounts No histochemical evidence of cell loss was demonstrable in parathyroid, submaxillary or adrenal glands, nor in heart, kidney or brain

In summary both types of transgenic animal, or the cells therefrom, were appratently normal until application of either ACG or 5FC, as appropriate After such application, either in vivo or in vitro the cells upon which sensitivity had been conferred were rapidly destroyed In addition cells from both transgenic animals were resistant to the cytotoxic effects of geneticin, whereas cells from non-transgenic controls were completely eradicated

Example 1 Proposed protocol for the production of a transgenic mouse bearing both positive and negative selectable markers The herpes simplex virus (HSV) thymidine kinase gene (tk) (operably linked to the tk promoter) and the bacterial neomycin phosphotransferase (neo) gene (operably linked to the SV40 early promoter) are cloned into the appropriate cloning sites of a plasmid vector

The plasmid vector is digested with restriction endonucleases and a fragment containing both the tk and neo selectable markers (along with the expression elements operably linked thereto) is isolated on an agarose gel

The fragment isolated on the gel is then purified and injected into male pronuclei of fertilized one-cell mouse eggs at a concentration of 1-2 ug/ml DNA in TE buffer (10mM Tπs, Ph 7 5, 0 2 mM EDTA) The eggs are those derived from a CBA x C57BU10 mating

The eggs which survive micro-injection are then transferred to pseudopregπant females as described e g in Wagner et al (1981 ) P N A S 78 5016, and allowed to develop to term At 7-14 days of age, each pup is analysed to determine whether the transgenes are present DNA is prepared from a section 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 probing with labelled tk and neo-specific probes The transgenic pups so identified are mated and their offspring also analysed to check for Meπdehan transfer of the transgenes

Example 2 Proposed protocol for the selective culture of mouse thyroid follicular cells Transgenic mice are prepared as descπbed in Example 1 , except that the neo gene is placed under the control of a thyroglobulin promoter (e g described by Chπstophe et al (1989) Mol Cell Endocπnol 64 5 Chπstophe et al (1987) Ann d'Endocπnol 48 111 , Ledent et al (1990), PNAS, 87 6176) The transgenic mice are sacrificed and the thyroid tissue removed and a primary culture prepared in the presence of antibiotic G418 This antibiotic kills eel's not expressing the neo gene, and results in the selective culturing within the primary (mixed cell) culture of thyroid follicular cells Example 3 Proposed protocol for the preparation of a rattine model of Parkinson's disease

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

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

Ganeiclovir is then administered by injection into the substantia nigra regions of the brain of the transgenic rats to specifically eliminate or deplete the dopaminergic neurones expressing the negative selectable tk marker, thus providing a rattine model of Parkinson's disease

Example 4 Proposed protocol for the preparation of a rattine model of Alzheimer's disease

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

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

Ganeiclovir is then administered by injection into the aapropπate region of the brains of the transgenic rats to specifically eliminate or deplete the acetylcholine- serotonin- and/or noradrenaline- neurones associated with the neo- and palaeocortex expressing the negative selectable tk marker, thus providing a rattine model of Alzheimer's disease

Table 1

Promoter Tissue/cell-tvoe Application Reference

Tyrosine Catecholamin- Parkinson's 1 hydroxylase -ergic neurones

TSH Thyroid cells Hypothyroidism 2 receptor B SF1 GABAergic Epilepsy 3 neurones

Human dopamine Noradrenaline Alzheimer's 4 b-hydroxylase neurones Thyroglobulin Thyroid cells Hypothyroidism 5

Serotonin 2 Glial cells in serotonin- Neurodegenerative 6 receptor ergic projection areas diseases

Mouse inter- bone cells and haemInflammatory leukin 4 atopoietic system processes C D4 receptor CD4 expressing AIDS T-lymphocytes human cnoline Acetylcholine Alzheimer's acetyltrans- neurones Motoneurone ferase disease

References

1 Stachowick et al (1994) Mol Brain Res 22 309 2 lkuyama and Nawata (1994) Jap J Clin Med 52(4) 962 3 Motejlek et al (1994) J Biol Chem 269 15265 4 Hoyle et al (1994) J Neurosci 14 2455 5 Pιchon et al (1994) Biochem J 298 537

6 Ding et al (1993) Mol Brain Res 20 181

7 Bruhn et al (1993) P N A S 90 9707

8 Nakayama et al (1993) Int Immunol 5 817

9 Li et al (1993) Neurochem Res 18 271

Table 2 Survival of lung fibroblast cells derived from control and transqenic rats, and effects of various drugs

Davs in culture

Genotype/drug 1 3 5 7 9 11

Control 100 100 100 100 100 100

TG/TK/neor 98 97 98 95 95 96

CD2/neor 92 93 92 98 105 98

Control + geneticin 9 101 85 23 5 2

TG/TK neor + geneticin 97 105 91 27 10 3

CD2/ neor + geneticin 91 94 91 93 107 105

Control -r 5FC 101 105 98 97 93 96

TG/TK neor + 5FC 98 96 95 92 92 93

CD2/neor + 5FC 94 5 3 3 4 4

Drugs were added at day 2 in culture Values were related to the number of cells found in control cultures without drug additions at various times after plating, and allowing for dilutions resulting from passaging Figures are the means of three separate determinations, the standard errors all being less than 15% of the mean

Table 3 Survival of thyroid cells derived from control and transgenic rats, and effects of various drugs

Days in culture

Genotype/drug 1 3 5 7 9 11

Control 100 100 100 100 100 100 TG/TK neor 91 95 93 92 101 99

CD2/neor 99 103 102 97 89 91

Control + geneticin 95 91 85 56 9 4

TG TK/neor + geneticin 104 105 98 88 93 98

CD2/ neor + geneticin 91 94 91 93 107 105 Control + ACG 94 97 98 91 92 102

TG/TK/neor + ACG 98 38 12 10 8 7

CD2/neor + ACG 98 90 93 93 97 88

Drugs were added at day 2 in culture. Values are related to the number of cells found in control cultures without drug additions at various times after plating, and allow for dilutions resulting from passaging. Figures are the means of three separate determinations, the standard errors all being less than 15% of the mean.

Claims

1. A graft (e.g. a xeπograft, allograft or autograft) comprising cells containing heterologous DNA compnsing a transgene encoding a negative selectable marker, wherein the negative selectable marker can be induced to activate programmed cell death or apoptosis.

2. Cells containing heterologous DNA comprising a transgene encoding a negative selectable marker, wherein tne negative selectable marker can be induced to activate programmed cell death or apootcsis. e.g. for use in transplantation therapy.

3. Transplantacle tissue or cells containing heterologous DNA comprising a transgene encoding a negative selectable marker, wherein the negative selectable marker can be induced to activate programmed cell death or apoptosis, e.g. for use in transplantation therapy.

4. A transgenic animal having ceils containing heterologous DNA compnsing a transgene encoding a negative selectable marker, wherein the negative selectable marker can be induced to actrv╬╡te programmed cell death or apoptosis.

5. The invention cf any one of the preceding claims wherein the negative selectable marker is:

(a) a tumour suppressor gene (e.g. p53);

(b) a ceil surface Fas receptor;

(c) an ιπterieukin-1 β converting enzyme (or activator thereof);

(d) c-myc or a omologue thereof; (e) retiπcciastoma gene;

(f) bax-cc-2 or a homologue thereof.

6. The invention of any one of the preceding claims wherein the cells:

(a) are essentially normal (e.g. functionally normal) but for the selectable phenotype aπsiπg from the transgene, optionally further comprising heterologous DNA comcπsiπg a transgene encoding a positive selectable marker and/or

(b) are Humanized; and/or

(c) are irπmunomodulated; and/or

(d) are neural, cardiac, eπdothelial, thyroid or pancreatic cells; and/or (e) are csπved from static cell populations, slowly replicating cell populations or expanding cell populations; and/or

(f) have a genotype which is essentially wild type but for the presence of the heterologous DNA and/or wherein that portion of the heterologous DNA which is expressed in the cells consists of the transgene encoding the negative selectable marKer taπd optionally a transgene encoding a positive selectable marker), the traπsceπeis) being operably linked to an expression element or elements.

7. The invention cf any one of the preceding claims which i s a vertebrate ( e . g . a mamma l or a non-human mammal, for example rat, rabbit, pig or mouse).

8. The invention cf any one of the preceding claims wherein at least the negative selectable marker is cperac.y linked to a regulatable expression element or elements, for example a tissue- or ceil-scecific expression element or elements or an inducible expression element (e.g. an ╬╣nc--c:^'c:s -.remoter).

9 The invention of claim 7 compnsing a positive and negative selectaoie mar<er, wherein each selectable marker is differentially regulated, each marker for example ceing linked to a different tissue- or cell-specific expression element or elements

10 The invention of any one of the preceding claims wherein at least one se'╬╡ctable marker (e g the positive selectaoie marker) is constitutively expressed.

11 The invention of any one of the preceding claims wherein the hetercicccus DNA further compπses a reporter transgene, for example β-galactosidase or luciferase.

12. The animal of claim 11 wnerein the reporter transgene is operably linkec to an expression element or elements which are subject to cell- or tissue-specific regulation.

13. The invention of any one of the preceding claims wherein: 5 (a) the positive selectable marker is selected from neomycin phcscπotransferase, hygromy n phosphotransferase, xanthiπeguanine phosphoπbosyl transferase, the Herpes simplex virus type 1 thymidine kinase, adenine phosphoπbosyltransferase aπα hypoxaπthine phosphoπbosyltransferase and or

(b) the expression element is selected frorrr 0 (I) promoters and/or enhancers which are specifically active in: (i) dopaminergic, serotcniπergic. GABAergic, cholinergic or peptidergic neurones and sub- populations thereof; (n) oiigcceπdrocytes, astrocytes and sub-populabcπs thereof; (ui) the endocπne glands, lungs, muscles, gonads, intestines, skeletal tissue or can cr parts thereof; (iv) epithelial, fibroblast, fat mast, mesenchymal or parenchymal cells; (v) canicular stages of 5 embryogenesis. and (vi) components of the blood system (e.g. T-lympπocyt--s. B-lymphocytes and macrophages), or

(II) promoters and/or enhancers which direct the transcnpπcn of genes for (i) neurotransmitter-soecmc receptors; (li) ion channels; (iii) receptors invcivec n ion channel gating and (iv) cytokiπes growth factors and hormones. 0

14 Tissue or cells deπved cr cultured from the transgenic animal of any cne of claims 4-13.

15. A method of cultuπng ceils and/or tissues in vitro, composing the stεos cf: (a) providing an animal as defined in any one of claims 4 to 13: 5 (b) generating a pπmary culture from ceils and/or tissue of the animal of step (a); and

(c) selectively growing the pπmary culture on the basis of the seleσsDle phenotype conferred by the genetic mateπal contained in the cells of the animal.

16 A method according to c:aιm 15 wherein at least one selectable marker s operabiy linked 40 to a tissue- or cell-specific expression element or elements, whereby in step (c) a particular cell/tissue type is selectively grown on the basis of the tissue- or cell-scec-fic expression therein of said at least cπe selectable marker, e g. to produce a homogeneous copulation of a particular class of ceils in pπmary culture.

45 17 A method according to claim 15 or claim 16 whereby step (c) reduces cr eliminates microbial (e g yeast and fungal) contamination of the tissue culture

18 Tissue or cells cultured oy the method of any one of claims 15 to 17 tre tissue or cells being for use e g as a tissue transplant, as a test subject in biochemical assays or as a 50 source of a protein of interest 19 Tissue or cells according to claim 14 or claim 18 for use in therapy

20 A method of selectively eliminating or depleting a particular tissue or cell type in an 5 organism comprising the steps of

(a) providing a transgenic animal according to any one of the preceding claims wherein the negative selectable marker is operably linked to an expression element (e g a promoter) specific for the tissue or cell type to be eliminated or depleted,

(b) eliminating or depleting that tissue or cell type on the basis of the expression 0 therein of the negative selectable marker

21 A method according to claim 20 for modelling disease-related cell/tissue loss or atrophy, wherein the tissue or cell type to be eliminated or depleted is that tissue or ceil type which is subject to disease-related elimination or atrophy 5

22 A transgenic animal according to any one of the preceding claims for use in the method of claim 20 or claim 21

23 An animal (for example a vertebrate, e g a mammal) in which a particular cell/ussue is 0 specifically eliminated or depleted, produced by the method of claim 20 or claim 21

24 An animal according to claim 23 which is a model of disease-related cell/tissue loss or atrophy

25 25 A method of screening compounds for pharmacological activity against a disease involving cell/tissue loss or atrophy, composing the steps of.

(a) providing a test model of the disease according to the method of claim 21;

(b) administering the compound to be tested to the test model of step (a);

(c) screening the compound to be tested on the basis of its effect on the test model. 30

26 A method according to claim 21 or claim 25 wherein the disease is

(a) Parkinson's disease and the tissue or cell-type to be eliminated or depleted comprises dopaminergic neurones in the substantia nigra,

(b) Huntington's chorea and the tissue or cell-type to be eliminated or depleted 35 compπses neural cells of the stπatum,

(c) Alzheimer's disease and the tissue or cell-type to be eliminated or depleted compπses acetylcholine-, serotonin- and/or noradrenaline- neurones associated with the neo- and palaeocortex,

(d) multiple sclerosis and the tissue or cell-type to be eliminated or depleted 40 comprises brain ohgodendrocytes

(e) immune disease and the cell-type to be eliminated or depleted compπses CD3, CD4 and/or CD8 cells, and

(f) AIDS and the cell-type to be eliminated or depleted compπses CD4 ce'ls

45 27 A method (e g an in vitro method) of determining the effect of a deficit in a first class of cells on the characteristics of a second class of cells in an organism, the method comprising the steps of

(a) providing a transgenic animal according to any one of the preceding claims having a first negative selectaoie marker operably linked to an expression e'ement specific for the

50 first class of cells and either (╬╣) a positive selectable marker operably linked to an expression etement specific for the second class of cells, or (ii) a second negative selectable marker linked to an expression element which directs the expression of the negative selectable marker in all cells of the organism except the second class of cells;

(b) administering a selective agent to the organism to induce a deficit in the first class of cells on the basis of the expression therein of the negative selectable marker;

(c) removing cells from the organism; and

(d) selectively cultuπng cells of the second class from those cells removed in step (c) on the basis of; (i) the expression therein of the positive selectable marker, or (ii) the lack of expression therein of the negative selectable marker.