Patent Application
20100138935
The present invention relates to a reporter gene construct, transfected cell lines including the construct and transgenic non-human animal models including the construct, the reporter gene construct incorporates nucleic acid sequences from a promoter region of a gene or set of genes whose expression is modified in response to external or internal changes in the cellular environment and in particular to stress conditions, the promoter being operatively linked to nucleic acid sequences chosen on the basis of the ease with which their transcription and/or translation products may be assayed. The reporter gene construct thereby provides a system capable of detecting intracellular conditions characteristic of biochemical stress or toxic conditions. In particular the present invention provides a reporter gene construct comprising, for example and without limitation, the cyclin-dependent kinase inhibitor 1 (p21) gene as the promoter region for the construct.
Contemporary methods of drug discovery, such as high-throughput screening of chemical libraries, identify large numbers of chemical entities with potential therapeutic effects. Only a fraction of these compounds will ever make useful drugs, however, because many of them have metabolic or toxicological characteristics that render them unsuitable for clinical use. Given the level of investment required to bring a potential new therapeutic drug to market, it is highly desirable that compounds possessing any adverse toxicological properties should be identified at an early stage, before significant development work has been undertaken.
A major problem with reliably screening large numbers of compounds for adverse toxicological characteristics is that methods currently available are either unsuitable for screening of large numbers of compounds or are too focused on specific mechanisms of toxicity to reliably predict the suitability of a given compound as a therapeutic drug. Thus, while in vivo pathological studies in experimental animals can give a very reliable indication of potential toxicity, the expense in terms of time and animal numbers required make such studies uneconomic for screening large numbers of compounds. The alternative to in vivo pathological studies is to employ in vitro methods of detecting known biochemical mechanisms. These are certainly amenable to high-throughput screening applications, but are limited in their ability to predict in vivo toxicity for a variety of reasons. These include the fact that: (i) only a selection of in vivo tissues can be represented cell culture systems; (ii) mechanisms of indirect toxicity seen in vivo are not replicated because effects of inter-organ interaction are absent in culture; (iii) the proliferation/quiescence state of cell lines or cells in primary culture is often different from their counterparts in vivo so that their responses are different; (iv) clonal selection of cell lines makes the results critically dependent on the particular cell line used.
A system that could monitor whole body indicators of toxicity in a manner that is economic for application to high-throughput screening would represent a very significant improvement over existing methods.
A cellular stress mechanism may be considered as the response of any gene or set of genes whose expression is modified in a cell, group of cells or tissue type in response to an external or internal change in the cellular environment that actually or potentially poses a threat to the normal functioning of the cell, neighbouring cells, tissues or the whole organism. Examples of changes in the cellular environment and genes whose expression may be transcriptionally modified as a result include:
1. Disturbances in the homeostatic state of DNA in the cell. These disturbances may include chemical alteration of nucleic acids or precursor nucleotides, inhibition of DNA synthesis and inhibition of DNA replication or damage to DNA. Genes whose expression may be altered in response to this type of disturbance include c-myc (Hoffman et al Oncogene 21 3414-3421), p21/WAF-1 (El-Diery Curr. Top. Microbiol. ImmunoL 227 121-137 (1998); El-Diery Cell Death Differ. 8 1066-1075 (2001); Dotto Biochim. Biophys. Acta 1471 43-56 (2000)), MDM2 (Alarcon-Vargas & Ronai Carcinogenesis 23 541-547 (2002); Deb & Front Bioscience 7 235-243 (2002)), Gadd45 (Sheikh et al Biochem. Pharmacol. 59 43-45 (2000)), FasL (Wajant Science 296 1635-1636 (2002)), GAHSP40 (Hamajima et al J. Cell. Biol. 84 401-407 (2002)), TRAIL-R2/DR5 (Wu et al Adv. Exp. Med. Biol. 465 143-151 (2000); El-Diery Cell Death Differ. 8 1066-1075 (2001)), BTG2/PC3 (Tirone et al J. Cell. Physiol. 187 155-165 (2001));
2. Changes in the oxidative status of the cell. Such changes may be brought about by e.g. accumulation of free radicals or hypoxia. Genes whose expression may be altered in response to changes of this type include MnSOD and/or CuZnSOD (Halliwell Free Radio. Res. 31 261-272 (1999); Gutteridge & Halliwell Ann. NY Acad. Sci. 899 136-147 (2000)), I□B (Ghosh & Karin Cell 109 Suppl., S81-96 (2002)), ATF4 (Hai & Hartman Gene 273 1-11 (2001)), xanthine oxidase (Pristos Chem. Biol. Interact. 129 195-208 (2000)), COX2 (Hinz & Brune J. Pharmacol. Exp. Ther. 300 376-375 (2002)), iNOS (Alderton et al Biochem. J. 357 593-615 (2001)), Ets-2 (Bartel et al Oncogene 19 6443-6454 (2000)), FasL/CD95L (Wajant Science 296 1635-1636 (2002)), GCS (Lu Curr. Top. Cell. Regul. 36 95-116 (2000); Soltaninassab et al J. Cell. Physiol. 182 163-170 (2000)), ORP150 (Ozawa et al Cancer Res. 61 4206-4213 (2001); Ozawa et al J. Biol. Chem. 274 6397-6404 (1999)).
3. Changes that cause hepatotoxic stress. Genes whose expression may be altered in response to hepatotoxic stress may include Lrg-21 (Drysdale et al Mol. Immunol. 33 989-998 (1996)), SOCS-2 and/or SOCS-3 (Tollet-Egnell et al Endocrinol. 140 3693-3704 (1999), PAI-1 (Fink et al Cell. Physiol. Biochem. 11 105-114 (2001)), GBP28/adiponectin (Yoda-Murakami et al Biochem. Biophys. Res. Commun. 285 372-377 (2001)), □-1 acid glycoprotein (Komori et al Biochem Pharmacol. 62 1391-1397 (2001)), metallothioneine I (Palmiter et al Mol. Cell. Biol. 13 5266-5275 (1993)), metallothioneine II (Schlager & Hart App. Toxicol. 20 395-405 (2000)), ATF3 (Hai & Hartman Gene 273 1-11 (2001)), IGFbp-3 (Popovici et al J. Clin. Endocrinol. Metab. 86 2653-2639 (2001)), VDGF (Ido et al Cancer Res. 61 3016-3021 (2001)) and HIF1 □Tacchini et al Biochem. Pharmacol. 63 139-148 (2002).
4. Stimuli that could trigger the cell to go into apoptosis. Examples of genes whose expression may modified by pro-apoptotic stress are Gadd 34 (Hollander et al J. Biol. Chem. 272 13731-13737 (1997)), GAHSP40 (Hamajima et al J. Cell. Biol. 84 401-407 (2002)), TRAIL-R2/DR5 (Wu et al Adv. Exp. Med. Biol. 465 143-151 (2000); El-Diery Cell Death Differ. 8 1066-1075 (2001)), c-fos (Teng Int. Rev. Cytol. 197 137-202 (2000)), CHOP/Gadd153 (Talukder et al Oncogene 21 4280-4300 (2002)), APAF-1 (Cecconi & Gruss Cell. Mol. Life Sci. 5 1688-1698 (2001)), Gadd45 (Sheikh et al Biochem. Pharmacol. 59 43-45 (2000( ), BTG2/PC3 (Tirone J. Cell. Physiol. 187 155-165 (2001)), Peg3/Pwl (Relaix et al Proc. Nat'l Acad. Sci. USA 97 2105-2110 (2000)), Siah 1a (Maeda et al FEBS Lett. 512 223-226 (2002)), S29 ribosomal protein (Khanna et al Biochem. Biophys. Res. Commun. 277 476-486 (2000)), FasL/CD95L (Wajant Science 296 1635-1636 (2002)), tissue tranglutaminase (Chen & Mehta Int. J. Cell. Biol. 31 817-836 (1999)), GRP78 (Rao et al FEBS Lett. 514 122-128 (2002)), Nur77/NGFI-B (Winoto Int. Arch. Allergy Immunol. 105 344-346 (1994)), CyclophilinD (Andreeva et al Int. J. Exp. Pathol. 80 305-315 (1999)), p73 (Yang et al Trends Genet. 18 90-95 (2002)) and Bak (Lutz Biochem. Soc. Trans. 28 51-56 (2000)).
5. Administration of chemicals, drugs or other xenobiotic agents. Examples of genes whose expression may be altered under such conditions are xenobiotic metabolising cytochrome p450 enzymes from the 2A, 2B, 2C, 2D, 2E, 2S, 3A, 4A and 4B gene families (Smith et al Xenobiotica 28 1129-1165 (1998); Honkaski & Negishi J. Biochem. Mol. Toxicol. 12 3-9 (1998); Raucy et al J. Pharmacol. Exp. Ther. 302 475-482 (2002); Quattrochi & Guzelian Drug Metab. Dispos. 29 615-622 (2001)).
6. Disease states either natural, modelled or induced. These diseases can be selected from but not limited to the list comprised of obesity, compromised immunity, degenerative neurological disorders, cancer, cardiovascular, inflammatory diseases, genetic diseases or metabolic disorders.
The p21 gene (also known variously as Cdkn-1a, WAF1, CIP1, SDI1 and MDA-6) encodes a protein whose cellular functions include inhibition of cyclin-dependent kinase and consequent cell cycle arrest and/or apoptosis (el-Deiry, W. S et al., Cell 75: 817-825, 1993; el-Deiry, W. S. et al., Cancer Res. 54: 1169-1174, 1994; Gartel, A. L. and Tyner, A. L, Mol. Cancer. Therapeutics 1: 639-649, 2002). Transcription of the p21 gene is subject to control by a variety of mechanisms associated with cellular stresses including regulation by the p53 tumour suppressor protein and other transcription factors such as Sp1, AP2, BRCA1, vitamin D3 receptor, retinoic acid receptor, C/EBPs, and STATs. Analysis of the p21 gene promoter region has identified a number of cis-acting sequence elements lying within 5 kilobases of the transcription start site through which these effects are mediated (Gartel, A. L. and Tyner, A. L, Exp. Cell Res. 246: 280-289, 1999).
Reporter gene systems incorporating sequences from the promoter region of the p21 gene have been used experimentally to demonstrate the ability of specific sequence elements to mediate the actions of specific transcription factors in regulating p21 expression in specific cell types (el-Deiry, W. S. et al., Cancer Res. 55: 2910-2919, 1995; Biggs, J. R. et al., J. Biol. Chem. 271: 901-906, 1996; Chin, Y. E. et al., Science 272: 719-722; Matsumura, I. et al., Mol. Cell. Biol. 17: 2933-2943, 1997; Zeng, Y. X. et al., Nat. Genet. 15: 78-82, 1997, Bellido, T. et al., J. Biol. Chem. 273: 21137-21144, 1998; Moustakas, A. and Kardassis, D., Proc. Natl. Acad. Sci. USA 95: 6733-6738, 1998; Biggs, J. R. and Kraft, A. S., J. Biol. Chem. 274: 36987-36994, 1999; Mitchell, K. O. and el-Deiry, W. S., Cell Growth Differ. 10: 223-230, 1999; Yang, W. L. et al., Mol. Cell. Biol. Res. Commun. 1: 125-131, 1999; Zhang, W. et al., J. Biol. Chem. 275: 18391-18398, 2000; Gartel, A. L. et al., Proc. Natl. Acad. Sci. USA 98: 4510-4515, 2001; Gartel, A. L. et al., Oncol. Res. 13: 405-408, 2003). However, there has been no prior account of a reporter gene system designed to provide readout of the entire range of transcriptional responses of the p21 gene or of its use for screening the effects of administered compounds in vivo.
It is known from the prior art to use Ho1-luc (heme oxygenase-luciferase) transgenic mice for in vivo screening for compounds as a marker of toxicity that induce luciferase expression following injection of a luciferein substrate (Malstrom et al 2004). However, Malstrom et al report that the luciferase signal induced by doxorubicin was from the intestine, kidneys, stomach, spleen, gonads and some liver and that “no treatment-related findings were observed in heart”. This is in sharp contrast to common general knowledge that the heart is a well-known site of doxorubicin toxicity (see Sun et al. 2001 and references therein). This observation suggests that the transgene used by Malstrom et al fails to detect potential toxicity in certain critical circumstances and so could give rise to false negatives.
Accordingly, a reporter system that could be regulated in a manner analogous to that of the endogenous gene and that would provide improved detection of cellular stress responses involving gene activation. We have found in the present invention that a reporter system incorporating for example the p21 gene or another stress responsive gene has the potential to provide an accurate and more sensitive means of detecting a wide variety of physiologically relevant cellular stresses and therefore satisfies the need for a means of early detection of many kinds of toxicity.
In the present invention we have developed a system based on the properties of stress responsive promoter sequences, for example and without limitation the p21 gene promoter. We have found surprisingly that construction of a reporter gene comprising a suitable array of promoter elements from, for example the p21 promoter, driving expression of a suitable readout gene can be used in a transfected cell lines or a transgenic animal to provide a rapid in vitro or in vivo assay of a wide variety of toxic mechanisms. The reporter transgenes of the present invention provide a system in which biochemical events associated with a variety of cellular stress mechanisms prognostic of toxicity can be examined conveniently in a whole animal.
According to a first aspect of the invention, there is provided a nucleic acid construct comprising (i) a nucleic acid sequence (the “Promoter Sequence”) of a promoter region of a gene or set of genes whose expression is modified in response to an adverse external or internal (extracellular or intracellular) change in the cellular environment and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) at least one nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript and providing a read-out in the form of an excretable protein and/or an agent capable of being detected histologically.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
It will be appreciated that the Reporter Sequence provides a read-out of the system into which the nucleic acid construct of the present invention has been incorporated so that nucleic acid sequence which acts as template for a defined RNA transcript may be in the form of an excretable protein, protein complexes or fragments, enzymes, enzymatic products or conjugates, primary, secondary or further metabolites and/or salts thereof, non-biological products that are released by direct or secondary effects on the expression of the reporter gene product, hormones or antibodies or the read-out may produce an agent which is not excretable from a cell but which is capable of being detected histologically, for example and without limitation LacZ products. It will also be appreciated that the nucleic acid constructs of the present invention may comprise more than one Reporter Sequence, for example it may contain two or more Reporter Sequences that express excretable read-out products or the nucleic acid may comprise a Reporter Sequence that expresses an agent capable of being detected histologically and an expressed excretable read-out product. The number of Reporter Sequences and variety thereof is not intended to limit the scope of the application.
Preferably, the promoter sequence of the gene has altered expression in response to any one or more of the following stress situations:
It will be appreciated that a luciferase reporter system is not desirable as a read-out system as luciferase detection requires the animals to receive a luciferin substrate. Luciferase expression will therefore be detectable only in organs and tissues accessible to injected luciferin and only before the luciferin has been cleared from the body. Moreover, there is a possibility that luciferin may affect the toxicity or other responses to administered test compounds.
In addition, using a luciferase reporter system, the animal under study must be anaesthetised or restrained. The stress and/or effects of anaesthesia itself may affect responses to administered test compounds. In the UK, legal limits on experimental procedures on living animals make repeated general anaesthesia or withholding of food and water for more than 2 hours extremely difficult to justify and would severely limit the application of the luciferase reporter readout system. Accordingly, in the present invention the Reporter Sequence is selected from the aforementioned examples with a view to minimizing background stress effects which could give rise to false readings.
Preferably, the promoter sequence of the construct of the present invention is selected from any one or more of the following genes comprising the group p21, metallothionein 1A, PUMA, Gclc, Cox2, Ki67, Stk6, Hsp, Hmox-1, Ho-1, PIG3 and SOD2.
In one embodiment of the invention, the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising up to 5000 base pairs of DNA from the region immediately 5′ to the transcription start site of a mammalian p21 gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
The Promoter Sequence may comprise sequences from within 5000 base pairs 5′ of the transcription start site of any mammalian p21 gene (also known as Cdkn1a, WAF-1, CIP-1, SDI1 and MDA-6), preferably from the human, rat or mouse gene. The promoter sequence may comprise a total of 166 to 5000 nucleotide base pairs comprising one or more individual selected sequence lengths each comprising one or more of the sequence intervals in the following list where the number ranges refer to the inclusive range of nucleotide base pair positions 5′ to the transcription start site: 0-5; 15-20; 64-69; 77-82; 93-143; 157-162; 688-696; 765-779; 1194-1212; 1256-1270; 1920-1928; 2553-2561; 4228-4236.
The Transcription Start Sequence may be provided by including part or all of the first non-coding exon of the p21 gene or a minimal eukaryote consensus promoter that will direct transcription by eukaryotic polymerases when associated with functional promoter elements or transcription factor binding sites, for example the PhCMV*-1 promoter (Furth et al., Proc. Nat. Acad. Sci. USA 91: 9302-9306, 1994).
The Reporter Sequence will be selected to provide a convenient read out of gene expression through assay of either the transcript or the encoded translation product polypeptide. Preferably, transcription of the Reporter Sequence may be conveniently detected by any one or more of the following:
(1) assay of the RNA transcript of the Reporter Sequence by, for instance:
The skilled person can select hybridization probes or PCR primer oligonucleotide sequences suitable for assay of any transcript to be assayed.
(2) assay of a polypeptide translation product of the Reporter Sequence by, for instance:
It will be appreciated that in some instance the reporter gene construct of the present invention may comprise the same Promoter Sequence but that the Reporter Sequence may be varied so that the construct may provide a number of different read-outs as a result of the same Promoter Sequence.
In a further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising a 26 kb upstream segment of a mammalian metallothioneien 1A gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising up to 16 kb of an upstream gene sequence and a 5 kb segment of downstream sequence of a mammalian PUMA gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising up to 16 kb of an upstream gene sequence of a mammalian Gclc gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising up to 3.5 kb sequence upstream from the transcriptional start site and a further 3 kb segment from the coding sequences of a mammalian cox 2 gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising up to 30 kb of an upstream gene sequence of the transcriptional start of a mammalian Ki67 gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising an E4TF1 motif (−87 to −78) and an Sp-1 site (−129 to −121) and optionally a tandem repressor element CDE/CHR (−53 to −49/−39 to −35) of a mammalian Stk6 gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising up to 5 kb of 5′ flanking region of a mammalian Hsp70 gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising up to 16.5 kb of upstream promoter and 8 kb of 3′ sequence of the mammalian Ho1 gene reporter locus and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
In a yet further embodiment of the invention the nucleic acid construct comprises (i) a nucleic acid sequence (the “Promoter Sequence”) comprising p53 recognition sequence repeats of a mammalian PIG3 gene and (ii) a nucleic acid sequence (the “Transcription Start Sequence”) comprising a transcriptional start site and (iii) a nucleic acid sequence (the “Reporter Sequence”) capable as acting as template for a defined RNA transcript.
According to a second aspect of the invention there is provided a nucleic acid construct comprising a Promoter Sequence, as defined under the first aspect of the present invention, operatively isolated from a Transcription Start Sequence and a Reporter Sequence, as defined under the first aspect of the present invention, by a nucleotide sequence (the “Isolator Sequence”) flanked by nucleic acid sequences recognised by a site specific recombinase, or by insertion such that it is inverted with respect to a Transcription Start Sequence and a Reporter Sequence, as defined under the first aspect of the present invention. The recombinase recognition sites are arranged in such a way that the Isolator Sequence is deleted or the inverted Promoter Sequence's orientation is reversed in the presence of the recombinase. The construct also comprises a nucleic acid sequence comprising a tissue-specific promoter operatively linked to a gene encoding the coding sequence for the site-specific recombinase.
This aspect allows for detecting Reporter Sequence expression in specified tissues only. By controlling the appropriate recombinase expression using a tissue-specific promoter, the inducible gene construct will only be viable in those tissues in which the promoter is active. For example, by driving recombinase activity from a liver-specific promoter, only the liver will contain re-arranged reporter construct, and hence will the only tissue in which Reporter Sequence expression can occur.
The recombination event producing an active Reporter Sequence expression unit may therefore only take place in tissues where the recombinase is expressed. In this way the Reporter Sequence may only be expressed in specified tissue types where expression of the recombinase results in a functional transcription unit comprised of the inducible promoter linked to the promoter. Site-specific recombinase systems know to perform such a function include the bacteriophage P1 cre-lox and the bacterial FLIP systems. The site-specific recombinase sequences may therefore be two loxP sites of bacteriophage P1
The use of site-specific recombination systems to generate precisely defined deletions in cultured mammalian cells has been demonstrated. Gu et al. (Cell 73: 1155-1164, 1993) describe how a deletion in the immunoglobulin switch region in mouse ES cells was generated between two copies of the bacteriophage P1 loxP site by transient expression of the Cre site-specific recombinase, leaving a single loxP site. Similarly, yeast FLP recombinase has been used to precisely delete a selectable marker defined by recombinase target sites in mouse erythroleukemia cells (Fiering et al., Proc. Nat. Acad. Sci. USA 90: 8469-8473, 1993). The Cre lox system is exemplified below, but other site-specific recombinase systems could be used.
A construct used in the Cre lox system will usually have the following three functional elements:
This construct can be eliminated from host cells or cell lines containing it by means of site-specific recombination between the two loxP sites mediated by Cre recombinase protein which can be introduced into the cells by lipofection (Baubonis et al., Nuc. Acids Res. 21: 2025-2029, 1993). Cells which have deleted DNA between the two loxP sites are selected for loss of the TK gene (or other negative selectable marker) by growth in medium containing the appropriate drug (ganciclovir in the case of TK).
According to the third aspect of the invention there is provided a host cell transfected with a nucleic acid construct according to any one of the previous aspects of the invention. The cell type is preferably of human or non-human mammalian origin but may also be of other animal, plant, yeast or bacterial origin.
According to the fourth aspect of the invention, there is provided a transgenic non-human animal in which the cells of the non-human animal express the protein encoded by the nucleic acid construct according to any one of the previous aspects of the invention. The transgenic animal is preferably a mouse but may be another mammalian species, for example another rodent, for instance a rat or a guinea pig, or another species such as rabbit, or a canine or feline, or an ungulate species such as ovine, porcine, equine, caprine, bovine, or a non-mammalian animal species, for instance an avian (such as poultry, for instance chicken or turkey).
In embodiments of the invention relating to the preparation of a transfected host cell or a transgenic non-human animal comprising the use of a nucleic acid construct as previously described, the cell or non-human animal may be subjected to further transgenesis, in which the transgenesis is the introduction of an additional gene or genes or protein-encoding nucleic acid sequence or sequences. The transgenesis may be transient or stable transfection of a cell or a cell line, an episomal expression system in a cell or a cell line, or preparation of a transgenic non-human animal by pronuclear microinjection, through recombination events in embryonic stem (ES) cells or by transfection of a cell whose nucleus is to be used as a donor nucleus in a nuclear transfer cloning procedure.
In embodiments of the invention relating to the preparation of a transfected host cell or a transgenic non-human animal comprising the use of a nucleic acid construct as previously described, the cell or non-human animal may be one that has undergone previous genetic modification resulting in the selective deletion or inhibition of expression of one or more endogenous genes. The said gene deletions or inhibitions may be effected by permanent deletion of selected sequences from the genome, conditional deletion of selected sequences, for instance by insertion of loxP sequences flanking the sequences to be deleted and subsequent conditional excision of said sequences to be deleted by constitutive or conditional expression of cre recombinase (U.S. Pat. No. 4,959,317) or selective interference with RNA transcripts, for instance by insertion into the genome of a DNA sequence directing expression of a short double-stranded RNA molecule with one strand complementary to the target gene (U.S. Pat. No. 6,573,099).
Introduction of the nucleic acid construct as previously described into a cell or non-human animal in which genetic modification resulting in the selective deletion or inhibition of expression of one or more endogenous genes has been effected, may be achieved by transient or stable transfection of a cell or a cell line in which deletion or inhibition of another gene has previously been effected, or by introduction of an episomal expression system into a cell or a cell line in which deletion or inhibition of another gene has previously been effected, or by preparation of a transgenic non-human animal by pronuclear microinjection, through recombination events in embryonic stem (ES) cells from an animal in which deletion or inhibition of another gene has previously been effected or by transfection of a cell from an animal in which deletion or inhibition of another gene has previously been effected whose nucleus is to be used as a donor nucleus in a nuclear transfer cloning procedure or by cross-breeding an animal as described under the fourth aspect of the present invention with another animal of the same species in which deletion or inhibition of another gene has previously been effected and subsequent genomic analysis of offspring of the crossing to identify individuals in which both the introduced nucleic acid construct of the present invention and the deletion or inhibition of the other selected gene are present, for instance by isolation of genomic DNA from a tissue sample and PCR analysis.
Methods of preparing a transgenic cell or cell line, or a transgenic non human animal, in which the method comprises transient or stable transfection of a cell or a cell line, expression of an episomal expression system in a cell or cell line, or pronuclear microinjection, recombination events in ES cells, or other cell line or by transfection of a cell line which may be differentiated down different developmental pathways and whose nucleus is to be used as the donor for nuclear transfer; wherein expression of an additional nucleic acid sequence or construct is used to screen for transfection or transgenesis in accordance with the first, second, third, or fourth aspects of the invention. Examples include use of selectable markers conferring resistance to antibiotics added to the growth medium of cells. For instance neomycin resistance marker conferring resistance to G418. Further examples involve detection using nucleic acid sequences that are of complementary sequence and which will hybridise with, or a component of, the nucleic acid sequence in accordance with the first, second, third, or fourth aspects of the invention. Examples would include Southern blot analysis, northern blot analysis and PCR.
According to the fifth aspect of the invention, there is provided the use of a nucleic acid construct in accordance with any one of the first, second, third, or fourth aspects of the invention for the detection of a gene activation event resulting from a change in altered metabolic status in a cell in vitro or in vivo.
The gene activation event may be the result of induction of toxicological stress, metabolic changes, disease that may or may not be the result of viral, bacterial, fungal or parasitic infection.
According to the sixth aspect of the invention there is provided the use of a nucleic acid construct comprising a Reporter Sequence, as defined under the first aspect of the present invention, wherein the transcription and/or translation products of said Reporter Sequence are heterologous to the cell in which the Reporter Sequence is expressed, for the detection of a gene activation event resulting from a change in altered metabolic status in a cell in vitro or in vivo.
The gene activation event may be the result of induction of toxicological stress, metabolic changes, disease that may or may not be the result of viral, bacterial, fungal or parasitic infection.
Uses in accordance with the fifth and sixth aspects of the invention also extend to the detection of disease states or characterisation of disease models in a cell, cell line or non human transgenic animal where a change in the gene expression profile within a target cell or tissue type is altered as a consequence of the disease. Diseases in the context of this aspect of the invention which are detectable under the methods disclosed may be defined as infectious disease, cancer, inflammatory disease, cardiovascular disease, metabolic disease, neurological disease and disease with a genetic basis.
An additional use in accordance with this aspect of the invention involves the growth of a transfected cell line in accordance with the third aspect in a suitable immunocompromised mouse strain (referred to as a xenograft), for example, the nude mouse, wherein an alteration in the expression of the reporter described in the first or second aspects of the invention may be used as a measure of altered metabolic status of the host as a result of toxicological stress, metabolic changes, disease with a genetic basis or disease that may or may not be the result of viral, bacterial, fungal or parasitic infection. The scope of this use may also be of use in monitoring the effects of exogenous chemicals or drugs on the expression of the reporter construct.
The fifth and sixth aspects of the invention extend to methods of detecting a gene activation event in vitro or in vivo.
In an embodiment according to the fifth aspect of the invention, the method comprises assaying a host cell stably transfected with a nucleic acid construct in accordance with any one of the first or second aspects of the invention, or a transgenic non-human animal according to the fourth aspect of the invention, in which the cell or animal is subjected to a gene activation event that is signalled by expression of a Reporter Sequence whose translation product is identified by means of an epitope tag peptide sequence.
In an embodiment according to the sixth aspect of the invention, the method comprises assaying a host cell stably transfected with a nucleic acid construct comprising a Reporter Sequence, wherein the transcription or translation products of said Reporter Sequence is heterologous to the cell in which it is expressed, or a transgenic non-human animal whose cells express such a construct, in which the cell or animal is subjected to a gene activation event that is signalled by expression of a Reporter Sequence whose translation product is identified by means of an epitope tag peptide sequence.
According to a yet further aspect of the invention there is provided a method of screening for, or monitoring of toxicologically induced stress in a cell or a cell line or a non-human animal, comprising the use of a cell, cell line or non human animal which has been transfected with or carries a nucleic acid construct as described above.
Preferably, the cell or a cell line or a non-human animal may comprise more than one gene construct according to the present invention in order that a variety of stress conditions may be monitored or screened simultaneously in a single cell or a cell line or a non-human animal. It will be appreciated that in this way a number of different read-outs may be obtained.
Alternatively in the method of the present invention a set or number of cells, cell lines or non human animals may be used simultaneously each comprising different gene construct(s) according to the present invention so that a variety of stress conditions may be screened or monitored simultaneously in a set or group. It will be appreciated that in this way a number of different read-outs may be obtained.
Toxicological stress may be defined as DNA damage, oxidative stress, post translational chemical modification of cellular proteins, chemical modification of cellular nucleic acids, apoptosis, cell cycle arrest, hyperplasia, immunological changes, effects consequent to changes in hormone levels or chemical modification of hormones, or other factors which could lead to cell damage.
Accordingly, there is also provided a method for screening and characterising viral, bacterial, fungal, and parasitic infection comprising the use of a cell, cell line or non human animal which has been transfected with or carries a nucleic acid construct as described above.
Accordingly, there is additionally provided a method for screening for cancer, inflammatory disease, cardiovascular disease, metabolic disease, neurological disease and disease with a genetic basis comprising the use of a cell, cell line or non human animal which has been transfected with or carries a nucleic acid construct as described above.
In these contexts the cell may be transiently transfected, maintaining the nucleic acid construct as described above episomally and temporarily. Alternatively cells are stably transfected whereby the nucleic acid construct is permanently and stably integrated into the transfected cells' chromosomal DNA.
Also in this context transgenic animal is defined as a non human transgenic animal with the nucleic acid construct as defined above preferably integrated into its genomic DNA in all or some of its cells.
Expression of the Reporter Sequence whose translation product is identified by means of an epitope tag peptide sequence in respect of the fifth aspect of the invention can be assayed for by measuring levels of the Reporter Sequence translation product in cell culture medium or purified or partially purified fractions thereof.
Skilled persons will appreciate how to choose Reporter Sequences whose translations products are known to be secreted into body fluids. For instance, a Reporter Sequence could be chosen to encode a lipocalin or the beta chain of human choriogonadotrophin, or secreted alkaline phosphatase any of which are secreted from the cells in which they are expressed and are eliminated into urine. Expression of a Reporter Sequence in accordance with the fourth aspect of the invention therefore can be assayed for by measuring levels of the translation product of said Reporter Sequence secreted into harvestable body fluids. In a preferred embodiment of the invention the body fluid will be urine, but may also be selected from the list including milk, saliva, tears, semen, blood and cerebrospinal fluid, or purified or partially purified fractions thereof.
Detection and quantification of Reporter Sequence translation products from cultured cells into tissue culture medium or transgenic non-human animal body fluid may be achieved using a number of methods known to those skilled in the art:
(i) The assay may be an ELISA whereby an antibody or antiserum containing a single or mixture of antibodies recognising translation product of the Reporter Sequence and is used as a capture antibody to coat a microtitre plate or other medium suitable for conducting the assay. The culture medium or body fluid containing the reporter gene product (analyte) is added to the microtitre plate to allow binding of the analyte. Addition of the same antibody or antiserum that has been conjugated to an enzyme, commonly horseradish peroxidase, is used as a second antibody. Addition of a suitable substrate, preferably one producing a colour product following conversion by the enzyme is used to quantify the analyte in proportion to how much second antibody conjugate has been bound.
(ii) Competitive ELISA. In an alternative form the tissue culture medium or the body fluid (analyte) sample containing the expressed Reporter Sequence translation product bound to a support suitable for conducting the assay. In a separate reaction a limited standard amount of antibody specifically recognising the reporter gene product is added to a separate aliquot of the same and allowed to bind. This is added to the analyte bound to the support to allow remaining free antibody to bind. A second, enzyme conjugated antibody against for example the Fc region of the first antibody is allowed to bind and the colorimetric readout can be used to quantify the analyte whereby the degree of colour change is inversely proportional to the level of analyte in the sample.
(iii) Western Blot Analysis
Transfected cell homogenates were prepared by incubation of cells in homogenization buffer (140 mM NaCl, 50 mM Tris-HCl pH7.5, 1 mM EDTA, 1% Triton-100) for 30 minutes on ice. Following a brief centrifugation to remove insoluble material the cleared supernatants were assayed for protein content. A volume equivalent to 40 μg cell extract and an equal volume of cell medium were subjected to SDS-PAGE and blotted onto nitrocellulose (Schleicher and Schuell, Dassel, Germany) membrane using a semi-dry blotting apparatus. (Bio-Rad, Richmond, Calif.). The membranes were blocked for 1 hour in blocking buffer (5% NFDM w/v in PBS) then incubated with myc mAb (Invitrogen Life Technologies, Carlsbad, Calif.) diluted in blocking buffer for 2 hours with continuous agitation. After a series of washes in PBST (PBS plus 0.05% Tween-20), the membrane was incubated in an anti-mouse antibody conjugated to HRP diluted in blocking buffer for one hour with agitation, and after another series of washes in PBST the HRP activity was developed using an ECL kit (Pierce, Rockford, Ill.) and captured on auto-radiographic film (Kodak).
(iv) Fluorescence polarisation. The antibody specifically recognising the Reporter Sequence translation product is conjugated with fluorescein and mixed with the analyte produced. This method quantifies the analyte by direct measurement of the amount of antibody-antigen complex present. This method may also be adapted to measure any protein-protein interaction.
2. Release of a labelled substrate. Detection of conversion of substrate due to enzymatic activity of the translation product of a Reporter Sequence. The nature of substrate conversion may or may not fall into one or more of the following event categories: Proteolysis, phosphorylation, acetylation or sulphation, methylation
3. Detection of multiple substrates. Where a multiplicity of Reporter Sequence translation products are used, methods suitable for detection of such events could include but not necessarily be limited to:
(i) Mass spectrometry
(ii) Nuclear magnetic resonance (NMR)
In a preferred embodiment of the invention there is provided a method of detecting a reporter gene activation event, comprising the steps of:
In step (1), the Reporter Sequence transcription and/or translation products may be heterologous to those already expressed in the cell in which the Reporter Sequence is expressed. In any case, the skilled person will appreciate how to engineer a Reporter Sequence with tagging sequences so that transcription and/or translation products of said Reporter Sequence will be heterologous to those already expressed in the cell in which the Reporter Sequence is expressed.
Methods and uses in accordance with the present invention offer significant advances in investigating any area in which modified gene expression plays a significant role. Such reporter genes will be of use in cells and transgenic animals to detect activity of the p21 gene. Specific applications include but are not restricted to:
Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.
The present invention will now be described with reference to the following examples which are present for the purposes of illustration only and should no be construed as being limited with respect to the invention. Reference in the application is also made to a number of drawings in which:
Two reporter constructs have been generated. Both contain a 4.5 kb fragment encompassing a contiguous segment of the mouse p21 gene promoter and upstream transcriptional control sequences. In one the p21 promoter segment is upstream from the coding sequence for a LacZ reporter gene and in the other it is upstream from the coding sequence for human chorionic gonadotrophin tagged with a unique epitope designed to facilitate detection by immunological assays. These constructs are referred to as pX3W (
The p21-LacZ and/or the p21-hCG construct is microinjected into fertilised mouse eggs which are implanted into pseudo-pregnant surrogate mothers. Pups are screened for the presence of the LacZ reporter gene by PCR analysis of tail biopsy tissue lysates. The sequence of the primers used for PCR screening are:
Samples are cycled as follows: 5.00 min @ 94° C., 35 cycles of (0.30 min @94° C., 0.30 min @ 61° C., 2.00 min @72° C.) 7.00 min @ 72° C., a @ 4° C.
Transgenic individuals identified by this method are bred with non transgenic animals to determine germline transmission of the transgene and establish transgenic mouse lines for analysis. All mice are maintained in a pathogen free environment.
Initial screening of transgenic offspring from founders generated and identified as described, is conducted by IP injection of a suitable compound known to induce activity of the wild type p21 gene; For example the topoisomerase II inhibitor etoposide is known to result in DNA damage. If dissolved in DMSO and injected intra-peritoneally at 40 mg/kg (LD50), subsequent examination of tissue sample sections by staining to detect LacZ reporter gene expression may be used to examine reporter gene expression characteristics of individual transgenic lines.
Targeted insertion of transgenes at the hypoxanthine phosphoribosyltransferase (Hprt) locus, on the mouse X chromosome was performed by Nucleis S.A., 60 Avenue Rockefeller, 69008 Lyon, France. The Hprt gene is a housekeeping gene, and as such is ubiquitously expressed in all tissues. This locus has been shown to exhibit no intrinsic enhancer activity in the majority of tissues and provides an exquisitely permissive chromatin environment that supports promoter dependent transgene expression (Farhadi et al., 2003; Heaney et al., 2004). Targeted insertion is achieved using BPES embryonic stem cells, in which the murine Hprt locus has been partially deleted, in combination with an Hprt targeting vector, pDEST, designed to restore Hprt gene function, and into which the transgene is cloned. The transgene is introduced into the pDEST targeting vector (Gateway® cloning, Invitrogen) by means of lambda phage-based site-specific recombination. In order to utilize this system, it is first necessary to create a low copy number plasmid that contains specific recombination sites (attL), a so-called Entry vector. This can be used initially to construct, then later to shuttle the transgene to the pDEST destination vector by recombination. The vector is generated by introducing the specific recombination sequences attL1 and attL2, separated by a spacer fragment, into the low copy number plasmid pACYC177, to create plasmid pLC15att. This vector is used for the subsequent cloning of reporter constructs.
Mice were housed on sawdust in solid-bottom, polypropylene cages and acclimatised for a period of 5 days before use. Temperature was maintained within a range of 19-23° C. and relative humidity within a range of 40-70%. There were 14-15 air changes per hour and twelve-hour periods of light were cycled with twelve-hour periods of darkness. Animals received RM1 diet (supplied by Special Diet Services Ltd., Stepfield, Witham, Essex, UK) ad libitum for the duration of each study. Drinking water, taken from the local supply and provided in bottles, was provided ad libitum. Animals were randomly allocated to groups, ear-numbered and weighed prior to the start of each experiment.
Each test compound was dissolved in the appropriate vehicle solution and administered by intraperitoneal injection (i.p.) at a dose volume of 10 ml/kg. Wherever possible, both naive (untreated) and vehicle-treated controls were used. If a test item whose toxicity in the mouse is poorly characterised was to be used, a dose-ranging experiment was conducted prior to the main experiment. The test item was administered to a single mouse which was monitored for any adverse effects 6-8 hours and 24 hours later. If the single dose ranging mouse tolerated the treatment, the remaining mice were administered the test item according to the protocol.
Terminal blood samples were taken by cardiac puncture into lithium/heparin-coated tubes for preparation of plasma. Tissues, as defined in each protocol, were routinely dissected as follows:
Two slices of tissue (˜2 mm thickness) were removed for histochemical analysis of LacZ expression according to methods described below.
Two further slices (˜2 mm thickness) were placed in 10% neutral buffered formalin (10% NBF) overnight then processed, sectioned and stained with haematoxylin and eosin (H&E).
The remaining tissue was placed in a cryovial and flash frozen in liquid nitrogen for RT-PCR and possible biochemical analysis.
Mouse brains were dissected so as to reveal the dentate gyrus as fully as possible. In the case of the kidneys, one kidney was cut in longitudinal section and the other in transverse section.
Following removal into tubes suitable for plasma preparation, venous blood samples were mixed on a roller for 10 minutes then cooled on ice. Red blood cells were removed by centrifugation at 2,000 rpm for 10 minutes at 8-10° C. The supernatant (plasma) was transferred to a second tube and stored at −70° C. until required for analysis.
Plasma samples were analysed for appropriate analytes on a Roche autoanalyser (Cobas Integra 400) according to the manufacturer's instructions. The routine markers measured included: (for liver damage) plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) and (for kidney damage) plasma creatinine and blood urea nitrogen (BUN).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay of mRNA
Analysis of the expression of endogenous genes was carried out by RT-PCR using the ABI Prism 7000 system. For RT-PCR analysis, frozen tissue samples were homogenised using a Polytron PT-mr2100 homogeniser in 1 ml TRI reagent (Sigma). Total RNA for each sample was isolated according to the manufacturer's instructions and RNA pellets stored at −70° C. under 75% ethanol. Each of the RNA pellets was resuspended in RNAase/DNAase free dH2O and quantified using a Genequant RNA/DNA calculator (Amersham). First strand cDNA synthesis was performed for each sample using 2 μg of total RNA and the Protoscript First Strand cDNA Synthesis Kit (New England Biolabs) according to the manufacturer's protocol. Additional steps to degrade RNA following cDNA synthesis were performed. Each sample was diluted to 50 μl with RNAase/DNAase-free dH2O and stored at −20° C.
In order to measure variation in p21 or Haemoxygenase-1 (HO-1) expression it was necessary to include an internal control. Amplification of β-Actin cDNA provides a standard to control for variations on RNA content between samples. The final reactions therefore consisted of two separate reactions (target gene and β-Actin cDNA amplification) performed simultaneously in a single tube. The PCR conditions used were 10 minutes at 95° C. followed by 40 cycles of denaturing for 15 seconds at 95° C. and annealing/extending for 1 minute at 60° C. The primers and probe for the p21 and HO-1 amplification reactions were obtained from ABI (Assay-On-Demand kit). These methods have been pre-validated for the standard conditions used on the ABI Prism 7000 system.
Tissues were washed with PBS and prefixed in 0.2% glutaraldehyde in PBS containing 0.1M MgCl2 and 5 mM EDTA, pH 7.3, for 4 hours at 4° C. on a shaker. After fixation, the tissues were transferred to 20% Sucrose solution and dehydrated overnight at 4° C. The next day they were embedded in cryo-M-bed medium on cork discs and lowered slowly into liquid nitrogen then transferred to −80° C. until sections were required.
Cryostat sample and chamber temperatures were set to −30° C. The sample to be sectioned was placed in the cryostat one hour prior to sectioning to allow equilibration at the appropriate temperature. Frozen sections (10 μm) were cut and placed on aminopropyltriethoxysilane (APES) coated slides.
Prior to staining, sections were incubated in 0.2% glutaraldehyde (as above) for 10 minutes at room temperature then washed twice (5 min each) in PBS containing 2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% % Nonidet-P40. Sections were then more of the same wash solution containing 1 mg/ml X-gal, 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide overnight at 37° C. then washed twice in PBS for 5 min, allowed to air dry and mounted in aqueous mounting medium (Hydromount). Slides were evaluated and photographed.
This method was as described by Campbell et al., 1996 and Campbell et al., 2005, with minor modifications.
Tissues were excised, immediately placed into chilled 4% paraformaldehyde containing 0.1% glutaraldehyde and 2 mM MgCl2 in PBS, and fixed in the above solution for 3 hours at 2-8° C. with gentle shaking. Fixed tissues were washed at 2-8° C. for 3×5 minutes with chilled PBS containing 2 mM MgCl2 and dehydrated overnight at 2-8° C. in PBS containing 2 mM MgCl2 and 30% (w/v) sucrose overnight The next day dehydrated tissues were embedded in OCT embedding medium using an isopentane/dry ice bath. Embedded tissues were stored at −70° C. until they could be cryosectioned.
Frozen sections were washed three times for 5 minutes in PBS containing 2 mM MgCl2 at 2-8° C. then 3×30 min in detergent wash (PBS containing 2 mM MgCl2 0.01% Nonidet P40 and 0.1% Sodium Deoxycholate) at 2-8° C. They were then stained overnight in PBS containing 2 mM MgCl2, 0.02% Nonidet P40, 0.1% Sodium Deoxycholate, 5 mM Potassium ferrocyanide, 5 mM Potassium ferricyanide, and 0.1% X-gal at 37° C. The next day, the slides were washed three times for 5 minutes in PBS containing 2 mM MgCl2 followed by three times for 5 minutes in PBS at room temperature. They were equilibrated with 100% ethanol (5 min) and counterstained with Eosin for 1 min at room temperature. Following dehydration and mounting they were evaluated and photographed.
Tissue specimens that had been prefixed in fresh 4% paraformaldehyde were stained immediately using the procedure for “Whole Mount X-Gal Histochemistry of Transgenic Animal Tissues” (see http://www.rodentia.com/wmc/docs/lacZ bible.html), summarised below) was followed, and the post-fixed tissues were transferred to 70% (v/v) ethanol.
Following fixation for one hour in 4% paraformaldehyde, tissues were rinsed three times, for thirty minutes each, with rinse buffer (100 mM sodium phosphate (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40) at room temperature. They were then stained in rinse buffer plus 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/ml X-gal (made up from 25 mg/ml stock in dimethylformamide (DMF), stored at −20° C.) for between 4 and 48 hours (typically overnight; about 90% of potential staining is believed to occur in the first 24 hours). Subsequently, the tissues were post-fixed overnight in 10% formalin at 4° C. and processed, sectioned and counterstaining with Neutral Red to reveal the nuclei.
Four sections of the promoter region of p21 (el-Deiry, W. S. et al., Cancer Res. 55: 2910-2919, 1995; Ensembl Mus-musculus transcript information ENSMUST00000023829) were individually amplified by polymerase chain reaction (PCR) from mouse genomic DNA (CB/4) using Herculase Taq (Stratagene) using the oligonucleotide primer pairs shown in
Also referred to as mt1, one of a family of small cysteine rich proteins with potent metal binding and redox capabilities, Mt1 plays an important role in Zinc homeostasis, and cellular response to heavy metal toxicity and oxidative damage. A complex array of transcription factor binding sites have been identified in the proximal promoter from −700 bp to the transcription start site. These include five MREs (MTF-1 binding), Sp1, ARE, USF1, AP-1 (plus AP-2, AP-4 in human). The five MREs clustered in the proximal promoter region (−153 to −43) participate in mediating transcriptional induction in response to heavy metals and to oxidative stress (Stuart et al., 1984 Proc Natl Acad Sci USA. 1984 December; 81(23):7318-22, Dalton et al., 1996 J Biol. Chem.; 271(42):26233-41). An overlapping ARE/USF recognition sequence at −100 is essential in maintaining basal expression and also plays a role in the activation of Mt1 expression in response to oxidative stress. MTF-1 and USF-1 have been reported to cooperate in essential regulation of Mt-1 expression in response to zinc in the developing embryo (Andrews et al., 2001 EMBO J. 20: 1114-112). The ARE mediates induction of GST-Ya subunit and the quinine reductase genes in response to H2O2 and redox cycling xenobiotics (Jaiswal, 1994 Biochem Pharmacol. 3; 48(3):439-44.). Reporters already constructed (LacZ and tagged hCG) for the mouse gene incorporate a 26 kb upstream segment of the gene, designed to encompass as yet uncharacterized upstream transcriptional modulatory elements. Others have made and studied mouse knockout models for mt1 and mt2: Mice homozygous for disruption of both alleles developed normally but were markedly more susceptible to hepatic poisoning with Cd than wild type (Masters et al., 1994 Proc. Nat. Acad. Sci. 91: 584-588). Beattie et al. (1998, Proc. Nat. Acad. Sci. 95: 358-363, 1998) also reported increased susceptibility to toxic stress, higher food intake and resultant obesity in mt1/mt2 double null mice. Amongst the stimuli eliciting elevated transcription of mt1 are heavy metals (e.g. cadmium) (Li et al., 1998 Nucl. Acids. Res. 26: 5182-5189), cobalt, zinc, H2O2, hydroxyl radical, heme analogues, ischemia, carbon tetrachloride, tin protoporphyrin, glucocorticoids, Phorbol esters, paraquat, diethyl and pyrrolidine dithiocarbamate, LPS, X-ray irradiation and inflammatory stress signals.
p53 up-regulated modulator of apoptosis, also referred to as BBC3. Exogenous expression of PUMA has been shown to result in extremely rapid apoptosis in cells in which it is expressed (Yu. J et al 2001). Its transcription, at least in part is elevated by binding of p53 to cognate promoter binding sites within the proximal promoter (p53 binding site BS1 at 230 bp upstream of transcriptional start and BS2 at 144 bp upstream of transcriptional start) are major p53 response elements characterized in the human promoter. Counterparts in the human promoter are sited at −410 bp and −375 bp respectively from the transcription start. However as the promoter region of PUMA is not well characterized reporters incorporate up to 16 kb of upstream gene sequence and 5 kb of downstream sequence so that as yet uncharacterized transcriptional control elements are included.
It is therefore envisaged that a PUMA gene reporter will be useful for detecting toxic insults that elicit apoptosis in a cell. Known inducers include DNA damaging agents such as 5FU, Adriamycin, Etoposide (Yu. J et al Proc. Nat. Acad. Sci. 100: 1931-1936, 2003, Yu. J et al Molec. Cell 7: 673-682, 2001, Nakano, K Molec. Cell 7: 683-694, 2001) and agents known to increase cellular level of p53 such as 4-hydroxytamoxifen (4-OHT) (Jia-wen Han et al 2001 PNAS 98 (20) 11318-11323).
Glutamate-cysteine ligase catalytic subunit (GCLC) along with the modulatory unit (GCLM) catalyses the first step in glutathione (GSH) biosynthesis which plays an essential role in protection of the cell against a range of toxic insults that cause accumulation of free radicals. Gclc transcription is know to be elevated by a range of chemical/xenobiotic agents, E.g. Chloroform, Cadmium Chloride, Acetaldehyde (Yang H. et al 2001 Biochem. J. (2001) 357: 447-455), Pyrrolidine dithiocarbamate (PDTC) in HepG2 and HEK293 cells (Erica L. Dahl et al 2001 Toxicological; Sciences 61, 265-272), Phenethyl isothiocyanate(PEITC) in HepG2 and HEK293 cells (Erica L. Dahl et al 2001 Toxicological; Sciences 61, 265-272), β-napthoflavone (β-NF) in HepG2 cells (Erica L. Dahl et al 2001) and cadmium chloride. A number of regions of the promoter are know to modulate gclc gene transcription; E.g. Yang et al (2001) conducted functional analysis of the promoter and describe three upstream flanking regions located between −595 to −111, −1108 to −705 (conferring positive regulation) and one between −705 to −595 (conferring negative regulation)
Within the proximal promoter at −416 to −316 NF-κB and AP-1 recognition sequences exist. Analysis of the human 5′ flanking region show it to contain an antioxidant-response element (ARE) and AP-1 and NF-κB binding sites. Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene is embryonic lethal when homozygous, and has been proposed as a model for moderate glutathione deficiency when heterozygous (Dalton et al., 2000 Biochem Biophys Res Commun. Dec 20; 279(2):324-9). It is therefore envisaged that reporters based on gclc will provide a reliable indicator of whether the test system (cells or transgenic animal) is being placed under oxidative stress. Reporters are designed such that coding region of the reporter gene is placed in the second exon, at the position of the natural first codon of the gclc locus, with 16 kb of upstream sequence providing known and putative uncharacterized transcription regulatory elements.
Murine Cyclooxygenase-2, also referred to as prostaglandin-endoperoxidase synthase 2 (Ptgs2) is responsible for catalysing the conversion of arachidonic acid to prostaglandins. it is induced in response to inflammatory signals e.g. IL-6 and is the main target for non-steroidal anti-inflammatory drugs such as aspirin. The literature reports a wide range of compounds that result in elevated expression of cox-2, E.g. Lipopolysaccharide, Cytokines, Mitogens: Superoxide, hydroxy radical, Arsenite, CCl4, 4-Hydroxy-2-nonenal, LPS, interleukin-1, tumor necrosis factor-alpha, PAF or interleukin-1beta, benzo[a]pyrene, ethanol and arachidonic acid, Phorbol 12-myristate 13-acetate, doxorubicin, Fe-NTA, deoxycholic acid12-O-tetradecanoylphorbol 13-acetate.
An expression cassette based on Cox2 potentially represents a good candidate reporter for any toxic insult eliciting an inflammatory response. The promoter region of the gene contains a complex array of known transcription factor binding sites such as NF-kB, (NF-kappaB-2 but not NF-kappaB-1), NF-IL6, ATF/CRE, E-Box, Pea3, SP1, AP-2, C/EBP, STAT3, STATI, TBP-response elements. The highly conserved 3′UTR is implicated in post-transcriptional control mediated at the level of mRNA stability, E.g. Phosphatidyl-inositol 3-kinase and AgC10 negatively regulate Cox-2 mRNA via the 3′UTR by inhibiting p38 MAPK required for the stability of the message. The reporter constructs therefore incorporate a ˜3.5 kb segment upstream from the transcription start site and a further ˜3 kb segment downstream from the coding sequences of cox2.
The name refers to the commercially available antibody recognizing a very large nuclear antigen highly expressed in proliferating cells (Schluter, C et al J. Cell Biol (1993) 123: 513-522). Expression of the antigen occurs during early G1, S, G2 and M phase of the cell cycle but is not detected in quiescent cells. Though the antibody is used widely in tumour pathology to detect proliferating cells in neoplastic disease states little is know about its function, though clearly its ubiquity in dividing cell populations suggests a cental role in cell division. The absolute correlation between high levels of Ki67 expression and cycling cell populations make it a potentially powerful tool on which to base a reporter gene for cell proliferation. As the gene and its regulation are as yet poorly characterized reporter gene design will utilize a large upstream segment of the gene (up to 30 kb upstream of the transcription start) that is designed to encompass as yet unidentified regulatory elements.
Serine/Threonine Kinase 6 (Aurora-family kinase 1), STK6, controls various mitotic events. The transcription of STK6 gene varies throughout the cell cycle and peaks during G2/M. The promoter of mouse STK6 has been characterized. An E4TF1 motif (−87 to −78) and an Sp-1 site (−129 to −121) have been identified as positive regulatory elements (Tanaka M et al (2002) JBC Vol. 277:10719-10726). A tandem repressor element, CDE/CHR (−53 to −49/−39 to −35), has been identified as a cell cycle regulatory element. By placing this region upstream of a reporter gene coding sequence a reporter cassette is potentially capable of signaling that the host cell population is about to undergo cell division in response to an external stimulus.
The induction of heat shock genes (hsp) in response to stress serves to protect against the initial insult and produce a state of resistance to subsequent stress. This protective role is attributed to an active participation in the folding of proteins, maintenance of proteins in their native folding states and the repair or promotion of the degradation of mis-folded protein. Hsp70 is involved in cell apotosis induced by a variety of stress stimuli. It therefore potentially provides the basis for reporters capable of signaling a range of stress stimuli. It is induced by UV radiation, heat shock, heavy metals and pathological (infections, fever, inflammation, malignancy) or physiological (growth factors, hormonal stimulation tissue development) stimuli. A 5.0 Kb 5′-flanking region of mouse Hsp70 gene contains regulatory motifs shown to respond to acute stress by mediating increased transcription of the hsp70 gene. Reporters have therefore been engineered on this basis; the reporter coding sequence is placed under the control of a sequence starting ˜5 kb upstream from the transcription start site of the hsp70 locus.
The HOZ reporter gene construct comprises regulatory sequences from the mouse haemoxygenase-1 (HO-1) gene operatively linked to LacZ, the coding sequence for β-galactosidase. HO-1 catalyses the initial reaction in the catabolism of haem to yield biliverdin and carbon monoxide and iron. Stimulation of HO-1 gene expression by most inducers is mediated at the level of transcription. Transcription is induced in response to oxidative stress, haem, heavy metals, heat shock and UV.
A number of previous studies have reported the derivation of clones that contained up to 14 bk of upstream sequences from the mouse HO-1 gene (Alam, 1994; Alam et al., 1994; Alam et al., 1995; Zhang et al., 2001).
Several enhancer regions have been identified from these studies. The more distal of these, the AB1 region, is located 10 kb upstream from the start site, indicating that at least 10 kb of promoter sequence would be required for correct induction by various inducing agents. A further enhancer region, SX2, in located at −4 kb, and several STAT binding sites at around −400 to −600 bp. In addition, an enhancer element has been identified within the introns of the human gene, which regulates the haem and cadmium response (Hill-Kapturczak et al., 2003).
We have used RedET recombination cloning (Zhang et al., 2000) to produce a HO-1-LacZ reporter transgene. A LacZ-SV40polyA minigene was engineered to contain homologous regions to exons 1 and 2 of the HO-1 gene. This minigene cassette also contains a bacterial Amp gene, at the 3′-end, which was used as a selectable marker during cloning. The minigene was then introduced, by homologous recombination, into a BAC clone containing the mouse HO-1 locus (clone RPCI-23 290L07, HGMP Resource Centre) substituting for the endogenous HO-1 exon1 and intron1 sequences. Thus, the HO-1 promoter would express LacZ from the ATG start codon, in place of the HO-1 protein. The correct position of the LacZ-SV40polyA minigene was confirmed by sequencing both the 5′- and 3′-junction regions. The HO-1 reporter locus, containing most of the HO-1 gene together with 16.5 kb of upstream promoter and 8 kb of 3′ sequence, was then recombined out from the BAC clone into the pACYC low copy number plasmid backbone.
Transient transfection of this construct into HEK293 cells, and induction with haemin resulted in an 11-fold increase in the LacZ labelling index over uninduced control cells.
Malstrom et al's HO-1 reporter uses 15 kb of HO-1 sequence upstream of the transcription start site. The HOZ reporter gene of the present invention uses 16.5 kb upstream and 8 kb downstream of the transcriptional start site. The reporter of the present invention is therefore likely to contain regulatory DNA sequences absent from that used by Malstrom et al and its expression is therefore more likely to be regulated in a manner analogous of that of the endogenous HO-1 gene and therefore a better detector of cellular stress responses involving HO-1 gene activation.
p53 induced gene 3 or PIG3 was initially identified by serial analysis of gene expression (SAGE) as one of a panel of 14 transcripts whose expression was elevated more than 10 fold in p53 expressing colorectal cancer cells versus control cells (p53 null) (Polyak et al., 1997 Nature Genet. 30: 315-320, 2002). Reactive oxygen species (ROS) are potent inducers of apoptosis; PIG 3 encodes a quinone oxidoreductase homologue that may have a role in the metabolism of ROS.
Contente et al., (2002 Nature Genet. 30: 315-320) demonstrated that PIG3 transcription is upregulated in direct response to binding of p53 to a pentanucleotide repeat sequence with in the PIG 3 promoter (TGYCC)n where Y=C or T. This sequence is both necessary and sufficient for transcriptional activation of the PIG3 promoter. On this basis a construct comprising a reporter gene sequence placed downstream from the proximal promoter and the upstream sequences from the PIG3 gene, that contain the p53 recognition sequence repeats, potentially provides a reporter for p53 induction in the cells or tissue in which the reporter gene is present
Manganese superoxide dismutase (MnSOD, SOD2) is a member of a family of metalloenzymes that catalyze the dismutation of the superoxide anion to H2O2. The SOD2 gene encodes an intramitochondrial free radical scavenging enzyme that is the first line of defense against superoxide produced as a byproduct of oxidative phosphorylation. It is a tumor necrosis factor (TNF)-inducible gene product. It plays an important role in the generation of the intracellular signalling molecule H2O2.
The promoter region of the SOD2 gene contains a number of well characterized transcription factor recognition sequences including SP-1; AP-1; AP-2; NF-κB; STAT3; C/EBP; Egr-1; TNFRE. Transcription factors SP-1 and AP-2 seem to have opposite roles in the transcriptional activity of the basal promoter. Whereas SP-1 plays a positive role, which is absolutely essential for transcription from the human MnSOD promoter, AP-2 appears to play a negative role in this process. An enhancer element is found in the promoter region of the human MnSOD gene. Several important enhancer elements are located in the second intron. The NF-kappa B site in the second intron is essential but not sufficient for high-level induction of MnSOD by cytokines. Although mutations in the regulatory elements may be partially responsible for the lack of induction of MnSOD in some cell types, differences in the degree of induction exist that cannot be accounted for by the defect in the DNA sequence. It is highly likely that this difference is due to the presence or absence of coactivator or suppressor proteins in the cells and may have a physiological role in the defense against oxidative stress.
The human MnSOD promoter lacks both a TATA and a CAAT box but possesses several GC motifs. It has been shown that the proximal promoter region (basal promoter) contains multiple Sp1 and AP-2 binding sites and that Sp1 is essential for the constitutive expression of the MnSOD gene. An Egr-1 binding site has been identified in the basal promoter of MnSOD. The basal promoter is responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the human hepatocarcinoma cell line HepG2. The contributions of these binding sites and the roles of the transcription factors Egr-1, AP-2, and Sp1 in the activation of hMnSOD transcription by TPA have been investigated by site-directed mutation analysis, Western blotting, and overexpression of transcription factors. The results showed that Sp1 plays a positive role for both basal and TPA-activated hMnSOD transcription, whereas overexpression of Egr-1 has a negative role in the basal promoter activity without any effect on TPA-mediated activation of hMnSOD.
Reporter constructs containing reporter gene sequence cloned between ˜11.8 kb of the 5′-promoter region and 18 kb of gene plus the 3′ region potentially provide a reporter for oxidative stress in the cells or tissue containing the reporter construct.
Injection of the pX3W construct into 431 fertilised mouse eggs as described resulted in a total of 96 offspring of which 12 were found to be transgenic by PCR analysis. Of these founder animals, six managed to successfully establish transgenic mouse lines. Of these, one line, WAZ44, was selected for detailed investigation based on the results of preliminary studies which indicated extremely low levels of basal transgene expression and transgene induction following toxic challenge.
Etoposide was selected as the compound for further evaluation with respect to LacZ induction in the WAZ44 mouse. Etoposide is a commonly used chemotherapeutic agent derived from epipodophyllotoxin whose mode of action as an anticancer agent involves inhibition of the enzyme topoisomerase II. Etoposide is a genotoxic carcinogen when administered chronically to mice. Its mode of action involves the induction of mitotic recombination leading to chromosomal rearrangements and loss of heterozygosity at key loci (Wijnhoven et al., 2003). Transgenic mice possessing an E. coli LacZ transgene which is only expressed after a DNA inversion involving the transgene have been used to demonstrate that etoposide causes significant induction of somatic intrachromosomal recombination events in vivo at doses between 0.05 and 50 mg/kg (Hooker et al., 2002, Sykes et al., 1999). Limited evidence does indicate that etoposide induces overt toxicity leading to chronic peritonitis and pleurisy in mice 3-4 weeks after i.p. administration of a toxic dose (Staehelin, 1976).
In experiments on WAZ44 mice, single doses of etoposide (of up to 40 mg/kg) were administered i.p. Animals were killed after 24 hours and tissues fixed and stained for LacZ expression. While little or no transgene expression was detected in control WAZ44 mice, animals that had received etoposide exhibited significant expression in the spleen, the thymus and in some regions of the brain.
Acrylamide, at a total dose of 150 mg/kg, induces neuropathy manifesting as partial paralysis approximately 7 days after the start of treatment. The dose may be administered as a single injection or divided; however, the effect depends upon the total dose, and since a single dose of 150 mg/kg is likely to be acutely toxic, a five-day coursed of injections of 30 mg/kg is preferred. Acrylamide intoxication is associated with both central and peripheral effects; the central effects include behavioural changes (increased milk-licking) which occur within two days of dosing, preceding peripheral changes such as reduced hind-limb grip and reduced locomotor activity by up to three weeks (Teal and Evans, 1982). In experiments on WAZ44 mice, five doses of acrylamide (30 mg/kg) were administered i.p.
A frozen section from the brain of an acrylamide-treated WAZ44 mouse was prepared and stained using Frozen Method 1. A dissecting microscope was used to view the slides and generate photomicrographs (a) transverse section of whole brain, original magnification ×10; (b) dentate gyrus and hippocampus, original magnification ×25 (c) cortex, original magnification ×63. An example of results obtained using acrylamide is illustrated in
To further investigate WAZ gene induction in the brain, we compared the effects of etoposide with those of mercuric chloride (HgCl2) which has been shown to induce p21 (Bartosiewicz et al., 2001), lipopolysaccharide (LPS) which has been shown to induce p21 in the hippocampus (Ring et al., 2002), and paracetamol which is known to cross the blood-brain barrier.
WAZ mice received single i.p. injections of etoposide (40 mg/kg); mercuric chloride (HgCl2, 8.5 mg/kg); lipopolysaccharide (LPS, 1.5 mg/kg) or paracetamol (300 mg/kg). WAZ mice received a single i.p. injection of each compound at a single dose level (see
The HOZ mouse contains a LacZ reporter gene driven by the HO-1 promoter described under Example 9. The HO-1-LacZ transgene was inserted into the murine Hprt gene (X chromosome), by homologous recombination, using the BPES cell line as described under ‘Targeted Transgenesis’.
Two ES cell clones were selected for injection into blastocysts and 4 male chimaeric mice were subsequently obtained from one of the clones. Three of the chimaeras were 100% ES derived and these male animals were bred to B6 females to produce 24 offspring. These animals were then used to examine the inducibility of the HOZ transgene reporter.
Preliminary characterisation of the HOZ transgene response to toxic insult was performed with cadmium chloride. Following oral administration of cadmium chloride, cadmium is absorbed via the proximal duodenum and transported to the liver, where it is deposited before being redistributed to the kidneys (Sorensen et al., 1993). Accordingly, cadmium causes acute hepatotoxicity followed by chronic nephrotoxicity. The nephrotoxic effect of cadmium is thought to be due to renal uptake of metallothionein-Cd which is synthesised in the liver following acute exposure and released into the circulation (Sendelbach and Klaassen, 1988).
The biochemical toxicity of cadmium involves a number of changes related to oxidative stress, including enhanced hepatic lipid peroxidation, glutathione depletion, upregulation of γ-glutamyl transpeptidase, and down-regulation of GSTs and CYPs (Andersen and Andersen, 1988, Dalvi and Robbins, 1978, Karmakar et al., 1999).
Initially, one HOZ mouse was given 10 mg/kg body weight cadmium chloride i.p. One control animal received the vehicle solution, isotonic saline, i.p. and an untreated animal was used as a second control. The animals were killed 24 hours after dosing and liver, kidney and brain were collected for H&E staining and lacZ histology.
Subsequently, a further experiment was carried out to extend these findings using a larger number of HOZ mice. For this experiment, both male and female HOZ mice were used and the effects of cadmium chloride in the dose range 2-8 mg/kg at 8 and 24 hours after administration were examined. The doses selected were based on previous experience and on literature reports that these doses cause HO-1 induction in mouse liver without inducing unacceptable toxicity (Aleksunes et al., 2005, Kenyon et al., 2005, Malstrom et al., 2004)
Sections of HOZ mouse liver and kidney were prepared and stained according to standard procedures (H&E). (a, b) Naive control; (c, d) Saline treated; (e, f) Cadmium chloride treated (10 mg/kg i.p. in saline). All images in
Sections of HOZ mouse liver were prepared and stained as described. (a, b) Naive control; (c, d) Saline treated; (e, f) Cadmium chloride treated (10 mg/kg i.p. in saline). The sections shown in images a, c and e were frozen according the Frozen Method 1 and stained according to Frozen Method 2 (original magnification ×200) and those shown in b, d and f were prepared and stained by Whole Mount Method (original magnification ×200). The livers of the naive and vehicle-treated control animals were devoid of LacZ staining (
With reference to
With reference to
When LacZ staining was evaluated in the kidney, punctate blue staining could be seen (
These results show induction of the HOZ transgene in kidney in response to cadmium chloride.
There was no evidence of LacZ expression in the brains of HOZ mice, with or without cadmium chloride treatment.
The data from Malstrom et al Ho1-luc transgenic mouse studies report that their luciferase signal in response to cadmium chloride was only “moderately” correlated with alanine aminotransferase (ALT) levels. In contrast, our results show HOZ transgene expression at lower doses of cadmium chloride before ALT elevation is evident. HOZ transgene expression is therefore a predictor of toxicity rather than merely being correlated with it. This evidence suggests that the system of the present invention is not only more accurate but is more sensitive than the prior art methods.
The effects of cadmium chloride on endogenous HO-1 expression were determined by RT-PCR assay of HO-1 mRNA levels in liver and kidney tissue from HOZ mice.
In animals that had received 10 mg/kg cadmium chloride, HO-1 mRNA levels in the kidney and liver after 24 hours were very substantially elevated compared to controls (
Previous reports on the induction of HO-1 by sodium (meta)arsenite indicated that HO-1 activity peaked at 8 hours following i.p. administration. We therefore examined the effects of sodium arsenite administration on HOZ transgene expression at 8 and 24 hours after dosing. Female HOZ mice received sodium arsenite at 13 mg/kg body weight in isotonic saline, i.p. and after either 8 hours or 24 hours, liver tissue was collected and stained for LacZ expression. These results indicated expression of the HOZ transgene at both time points (
Two plasma enzyme markers, ALT and AST, were used as markers of hepatotoxicity in HOZ mice treated with cadmium chloride or sodium arsenite. The results of this analysis for HOZ mice treated with 10 mg/kg cadmium chloride are shown in Table 1 and for HOZ mice treated with 2-8 mg/kg cadmium chloride or 13 mg/kg sodium arsenite in Tables 2 and 3.
Female HOZ mice exhibited a marked and rapid response to cadmium chloride at 8 mg/kg. The increase in plasma ALT was 170-fold after 8 hours and 275-fold after 24 hours, while the corresponding increases for AST were 237-fold after 8 hours and 97-fold after 24 hours. The dose response was steep: the increase in plasma ALT in response to a 4 mg/kg dose of cadmium chloride was only 2.4-fold after 8 hours and there was no increase above 2-fold in ALT after 8 hours, while no increase in plasma AST was observed at either time point. The response of male HOZ mice to cadmium chloride was slower and more muted, with a 7.9-fold increase in ALT and a 3.8-fold increase in AST 24 hours after treatment with 8 mg/kg but otherwise no increase above 2-fold. HOZ mice did not exhibit a hepatotoxic response to sodium arsenite.
Nephrotoxicity was estimated by measuring the blood urea nitrogen (BUN) levels as shown in Table 4. Female mice exhibited mild nephrotoxicity, as measured by BUN, in response to cadmium chloride at 8 mg/kg. The increase in BUN was 6-fold at 8 hours and 44-fold after 24 hours. No nephrotoxicity, as measured using this marker, was observed in male mice.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0415963.8 | Jul 2004 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB05/02779 | 7/15/2005 | WO | 00 | 8/6/2009 |
