Patent Application
20100132055
The present invention relates to a non-human animal model, cell and tissue cultures derived therefrom, which do not produce or produce only suboptimal levels of one or more functional sestrins and in addition do not produce or do only produce suboptimal levels of latent transforming growth factor β binding protein 4 (ltbp4). Furthermore, the present invention relates to a method for selecting agents for treating the pulmonary emphysema and/or the colorectal cancer exhibited by utilizing the animal model, cell or tissue culture of the invention. The animal model, cell or tissue culture is suitable for preclinical testing of efficacy, toxicity and bioavailability of potential agents.
Chronic obstructive lung disease with pulmonary emphysema (COPD) is a highly prevalent disease that has a large impact on quality of life for patients and their families and kills millions of people worldwide. While the major risk factor for COPD is the inhalation of toxic gases and particles that are primarily but not exclusively generated in tobacco smoke, primary (genetic) airway abnormalities are also involved as only a fraction of smokers develop COPD. COPD is associated with major healthcare costs, largely due to expensive treatments such as long-term oxygen therapy and hospital admissions, as well as indirect costs including loss of working capacity. Moreover, treatment of COPD is limited to symptoms as no pharmacologic intervention has been shown so far to modify the natural history of the disease (Vestbo J. and Hogg, J. C. Thorax. 61:86-8, 2006; Fabbri L. M., et al. Am J Respir Crit Care Med. 173:1056-65, 2006).
Colorectal cancer is one of the most common neoplasms in the Western hemisphere and represents a major public health challenge despite progress in detection and therapy. The overall mortality at 5 years is about 40%. While early stage disease can be cured by surgery, only palliative treatment is available for patients with non-resectable, metastatic cancer whose survival rate is between 7-24 months (Shaheen N. J., et al. Am J. Gastroenterol. 2006).
From the above in becomes apparent that new pharmaceutical compounds for the treatment of both conditions are highly desirable and could be identified with the animal model of the invention, which exhibits both pulmonary emphysema and colorectal cancer as a result of loss of function mutations in the sestrin 2- and the latent transforming growth factor beta binding protein 4 genes.
Transforming growth factor-βs (TGF-βs) belong to a protein superfamily whose members control cell growth and differentiation in a variety of adult tissues and are involved in a wide range of immune and inflammatory responses (Shi Y. and Massague, J. Cell. 113:685-700, 2003; Sheppard D. Proc Am Thorac Soc. 3:413-7, 2006). Most cells secrete TGF-β as a functionally inactive, latent cytokine complexed to its latency associated propeptide (LAP), which interacts with its receptors only after activation by proteolytic cleavage. There are two known, structurally different latent complexes of TGF-β. The small latent complex consists of a mature TGF-β dimer bound to the N-terminal portion of the TGF-β-LAP propeptide. The large latent complex contains, in addition to TGF-β-LAP, one of the three latent TGF-β binding proteins (ltbps 1, 3 and 4). Unless displaced from the ltbps after secretion, TGF-β is usually deposited in the extracellular matrix (ECM) as a high molecular weight complex with ltbp and LAP (Annes J. P., et al. J Cell Sci. 116:217-24, 2003). All 4 existing isoforms of ltbp (ltbp 1-4) have been mutationally inactivated in the mouse (Dabovic B., et al. J Cell Biol. 56:227-232, 2002; Sterner-Kock A., et al. Genes Dev. 16:2264-2273, 2002; Shipley J. M., et al. Mol Cell Biol. 20:4879-87, 2000). While the phenotypes of the knock out mice varied among isoforms, both ltbp3 and ltbp4 mutants develop pulmonary emphysema due to defects in TGF-β signaling. However, in contrast to the emphysema developing mutant ltbp3 mice, which is relatively mild and strictly developmental (Dabovic B., et al. J Cell Biol. 56:227-232, 2002), emphysemas in mutant ltbp4 (ltbp4−/−) mice worsen with age and progressively acquire the features of late onset emphysemas that develop in the context of COPD in humans (Sterner-Kock A., et al. Genes Dev. 16:2264-2273, 2002; WO 03/015505). The potential relevance of ltbp4 in the pathogenesis of COPD has been recently underscored by a clinical study reporting a highly significant correlation between specific COPD symptoms and a single nucleotide polymorphism (SNP) in ltbp4 (Hersh C. P., et al. Am J Respir Crit Care Med. 173:977-84, 2006). In addition to the emphysema, mutant ltbp4 mice develop invasive colorectal cancer (Sterner-Kock A., et al. Genes Dev. 16:2264-2273, 2002; WO 03/015505). A link between ltbp4 expression and cancer progression has also been recently reported in a clinical study of patients with colon cancer (Bertucci F., et al. Oncogene. 23:1377-91, 2004).
Sestrin 2 belongs to a family of highly conserved proteins initially discovered as p53 inducible proteins (Velasco-Miguel S., et al. Oncogene. 18:127-37, 1999; Peeters H., et al. Hum Genet. 112:573-80, 2003; Budanov A. V., et al. Oncogene. 21:6017-31, 2002; WO 00/12525; US 2002/0103353). Mammalian cells express three isoforms referred to as sestrin 1 (sestrin 1; also known as PA26), sestrin 2 (sestrin 2; also known as Hi95) and sestrin 3 (
In eukaryotic cells Prxs are believed to be both antioxidants protecting against stress and regulators of ROS-mediated signaling (Wood Z. A., et al. Science. 300:650-3, 2003) It has been known for some time that receptor/ligand interactions generate bursts of ROS, which act as second messengers in signal transduction pathways, including the TGF-β pathway (Bae Y. S., et al. J Biol Chem. 272:217-21, 1997; Lo Y. Y. and Cruz, T. F. J Biol Chem. 270:11727-30, 1995; Mills E. M., et al. J Biol Chem. 273:22165-8, 1998; Sundaresan M., et al. Science. 270:296-9, 1995; Thannickal V. J., et al. J Biol Chem. 273:23611-5, 1998; Thannickal V. J., et al. Faseb J. 14:1741-8, 2000). Defective TGF-β signaling has been shown to be involved in the pathology of pulmonary emphysema and colorectal cancer in both animal models (including WO 03/015505 A3) and men (Morris D. G., et al. Nature. 422:169-73, 2003; Neptune E. R., et al. Nat Genet. 33:407-11, 2003; Sterner-Kock A., et al. Genes Dev. 16:2264-2273, 2002; Massague J., et al. Cell. 103:295-309, 2000; Zhu Y., et al. Cell. 94:703-14, 1998).
The association between ROS and TGF-β is complex and occurs at different levels of the signaling cascade. First, ROS have been shown to activate latent TGF-β1 both in vitro and in vivo (Annes J. P., et al. J Cell Sci. 116:217-24, 2003; Barcellos-Hoff M. H. and Dix, T. A. Mol Endocrinol. 10:1077-83, 1996). In line with this, Fatma et al. have recently reported that lens epithelial cells derived from Prx6 knock out mice are highly susceptible to oxidative stress and develop a phenotype indistinguishable from TGF-β stimulation. They could attribute this phenotype to a ROS mediated activation of latent TGF-β, which was readily reversible by antioxidants (Fatma N., et al. Cell Death Differ. 12:734-50, 2005). Second, TGF-β requires ROS for the induction of various target genes such as PAI-1, CTGF and extracellular matrix genes (Jiang Z., et al. Biochem Biophys Res Commun. 309:961-6, 2003; Park S. K., et al. Biochem Biophys Res Commun. 284:966-71, 2001). Recent studies have shown that TGF-β induced smad2,3 phosphorylation is also partly dependent on ROS (Cucoranu I., et al. Circ Res. 97:900-7, 2005) presumably via the inactivation of a dedicated phosphatase. It has been known for some time that phosphatases are susceptible to inactivation by ROS (Seo J. H., et al. Mol Biol Cell. 16:348-57, 2005; Chiarugi P. and Cirri, P. Trends Biochem Sci. 28:509-14, 2003) and a smad2,3 phosphatase (PPM1A) capable of terminating TGF-β signaling has been recently discovered (Lin X., et al. Cell. 125:915-28, 2006. Third, TGF-β itself induces superoxide production by activating NADPH oxidases presumably by the transcriptional upregulation of Nox4 (Sturrock A., et al. Am J Physiol Lung Cell Mol Physiol. 290:L661-L673, 2006).
It was now found that the genetic ablation of a sestrin, notably sestrin 2, from the animal model (ltbp4−/− mice model) described in WO 03/015505 resulted in a significant recovery of the disease conditions (see examples), indicating that this family of proteins could provide molecular targets for the treatment of COPD and colorectal cancer. It is believed that ablation of sestrin 2 in the animal model of WO 03/015505, reactivated TGF-β signaling, presumably by increasing intracellular second messenger ROS levels. This in turn significantly improved both disease conditions as illustrated by the examples, suggesting that pulmonary emphysema and colon cancer can be treated by antagonizing sestrin function. The present invention thus relates to
(1) a non-human animal model, which does not produce one or more functional sestrins or produces subotpimal levels of one or more sestrins, preferably sestrin 2 and in addition does not produce latent transforming growth factor β binding protein 4 (hereinafter “ltbp4”) or produces subotpimal levels of ltbp4;
(2) a non-human animal model, which does not express one or more functional sestrins or expresses subotpimal levels of one or more sestrins;
(3) a cell or tissue culture isolated from the animal models as defined in (1) or (2) above;
(4) a method for preparing the non-human animal model of (1) above, which comprises disrupting the sestrin and/or ltbp4 gene in a germ cell of a starting non-human animal;
(5) a method for selecting an agent for treating a symptom occurring in the non-human animal model of (1) above comprising:
(i) applying one or more agents to be tested to said animal model,
(ii) determining whether one or more symptoms occurring in said animal model have changed as a result of application of said agent(s);
(6) a method selecting an agent that interferes with ROS production and TGF-β signaling comprising:
(i) applying one or more agents to be tested to the to the cell or tissue culture of (3) above,
(ii) determining whether cellular ROS levels and TGF-β signaling have changed as a result of the application of said agents or agents;
(7) a method to analyze whether cancer and/or pulmonary emphysema is caused by differential ltbp4 and sestrin gene or protein expression or expression level or by a defect in the ltbp4 and sestrin gene comprising:
(i) characterizing the ltbp4 and sestrin gene or protein expression or expression level or ltbp4 and sestrin gene allele status of an individual having cancer or pulmonary emphysema,
(ii) characterizing the ltbp4 and sestrin gene or protein expression or expression level or ltbp4 and sestrin gene allele status of a control individual, a difference in the ltbp4 and sestrin gene or protein expression or expression level or ltbp4 and sestrin gene allele status indicating that cancer and/or pulmonary emphysema and/or cardiomyopathy is linked to differential ltbp4 and sestrin gene or protein expression or expression level or a defect in the ltbp4 and sestrin genes;
(8) a method for diagnosing cancer and/or pulmonary emphysema and/or cardiomyopathy comprising:
(i) characterizing the ltbp4 and sestrin gene or protein expression or expression level or ltbp4 and sestrin gene allele status of an individual,
(ii) characterizing the ltbp4 and sestrin gene or protein expression or expression level or ltbp4 and sestrin gene allele status of a control individual, a difference in the LTBP-4 gene or protein expression or expression level or ltbp4 and sestrin gene allele status indicating the presence of cancer and/or pulmonary emphysema and/or cardiomyopathy in said individual;
(9) a kit for diagnosing pulmonary emphysema and/or cancer comprising means to detect the ltbp4 and sestrin gene or protein expression or expression level or the ltbp4 and sestrin gene allele status of an individual and a control individual; and
(10) a kit for performing the method of (6) above, said kit comprising the cell or tissue culture of (3) above.
The kits of (9) and (10) above may further contain instructions for performing the methods, in particular for detecting the ltbp4 and sestrin gene or protein expression or expression level or ltbp4 and sestrin gene allele status.
In conclusion, the present invention provides an animal model of human disease that reveals some crucial functions of the sestrin proteins. In view of these functions, the animal model of the invention can be used to develop novel treatments for pulmonary emphysema and cancer that target sestrin proteins and their role in ROS metabolism and TGF-β signaling.
The particular terms and abbreviations utilized to define the present invention are further defined in the following.
The terms “does not produce functional sestrins” and “does not produce functional ltbp4” denote the lack of effective sestrin and ltbp4 expression, respectively. A “lack of effective sestrin and ltbp4 expression” also includes the expression of non-functional (i.e. truncated) sestrins and ltbp4 proteins which are not exerting the function of the native protein.
The term “produces suboptimal levels of sestrins” and “produces suboptimal levels of ltbp4” encompasses the translation level of sestrin and ltbp4 proteins, which are insufficient to exert their function. Preferably, the level is reduced at least by 50%, more preferably by 70% and most preferably by 100%.
The term “homozygous disruption” relates to an identical mutation in both alleles of a gene.
The term “heterozygous disruption” relates to a mutation in only one allele of gene. The term “mutation” refers to a change of one or more nucleotide pairs of a DNA molecule.
The term “insertion” is directed to a mutation identified by the presence of one or more additional base pairs in the DNA. The term “deletion” relates to a mutation generated by removal of a sequence of DNA (one or more base pairs), the regions on either side being joined together. The term “substitution mutation” is directed to a nucleotide exchange. The substitution mutation can result in an amino acid change or can introduce a premature translation stop codon. Furthermore, a substitution mutation can affect splicing or expression of the gene when occurring at sites necessary for splicing or gene regulation.
The term “gene targeting” relates to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a cell and that fragment recombines with homologous sequences in the genome. The term “gene trap integration” is directed to insertion of a vector, which comprises a reporter gene and which is activated upon insertion into an active transcription unit of the genome.
The term “mutagenesis” denotes a chemical or physical treatment that changes nucleotides in the genome of an organism. An example of a chemical mutagenesis is N-ethyl-N-nitrosurea (ENU) mutagenesis.
The term “exon” encompasses a segment of a gene, which is decoded to give a mRNA product. Individual exons may contain protein coding DNA and/or noncoding DNA. The term “intron” denotes non-coding DNA, which separates neighboring exons in a gene. During gene expression, introns like exons are transcribed into RNA but the transcribed intron sequences are subsequently removed by RNA splicing and are not present in mRNA. The term “regulatory region” relates to the nucleotide sequence, which comprises regions that are necessary for the regulation of gene transcription. These regions comprise, for example, promoters and enhancers and they can be located in 5′ untranslated regions, exons, introns and 3′UTRs. The term “splice site” encompasses the nucleotides at the beginning and the end of the intron that are required for the joining of two exons by removing the intercalated intron during primary transcript processing to functional mRNA.
The term “pulmonary emphysema” denotes a symptom of chronic obstructive lung disease (COPD) characterized by a size increase beyond normal of air spaces distal to the terminal bronchioles and inflammatory infiltrates.
The term “cardiomyopathy” designates a primary non-inflammatory disease of the heart muscle, which is the result of pulmonary hypertension that complicates COPD. The term “cancer” refers to an uncontrolled proliferation of epithelial cells lining the colonic crypts.
“ROS metabolism” refers to the production and neutralization (reduction) of intracellular reactive oxygen species such as hydrogen peroxide and oxygen anions.
The term “profibrotic changes” refers to an increased tissue deposition of collagen associated with a multiplication of fibroblasts.
The term “selecting an agent for treating a symptom” encompasses choosing a composition for management of the condition.
The term “application of one or more agents” relates to administering single compounds or compound combinations orally, by inhalation, parenterally, e.g. intravenously, subcutaneously, intraperitoneally or intramuscularly, or topically, e.g. ophtalmically, vaginally, rectally or intranasally.
The invention is hereinafter described in more detail by referring to the accompanying Figures and Examples.
In a preferred embodiment of aspect (1) of the invention, the genome of the animal comprises a homozygous disruption of the sesn2 and ltbp4 genes. Preferably this homozygous disruption has been generated by a mutation and this mutation can be an insertion, deletion or a substitution mutation. Furthermore, preferably said mutation is generated by gene targeting, gene trapping or chemical mutagenesis and it has occurred in an exon, intron, regulatory region or splice site of the sestrin 2 gene, preferably into the last intron (i.w. the 9th intron) of the sestrin 2 gene, and in an exon, intron, regulatory region or splice site of the ltbp4 gene, preferably in the 5th intron of ltbp4. It is also preferred that the mutation sites rise to an expression of a detectable reporter gene such as fluorescent proteins (such as GFP and its derivatives), enzymes (such as LacZ) or selection markers (such as βgeo), or the like. Particularly preferred is that
In a further preferred embodiment said animal comprises a heterozygous disruption of the sens2 gene and a homozygous disruption of ltbp4 gene or homozygous disruption of the sestrin 2 gene and a heterozygous disruption of the ltbp4 gene.
In another aspect of the invention the animal model exhibits pulmonary emphysema and/or cardiomyopathy and/or colorectal cancer. Furthermore, the non-human animal is preferably a non-human mammal, more preferably a rodent including mouse and rat.
In a further preferred embodiment the animal model exhibits defects in ROS metabolism and/or profibrotic changes in one or more major organs, preferably in the lung or colon.
In a further preferred embodiment the animal model develops symptoms that are less severe than those exhibited by the animal model disclosed in WO 03/015505 A3.
Embodiment (3) of the invention relates to a cell or tissue culture isolated from a non-human animal model (1) or (2). Preferably, the cell is derived from the lung or colon.
In a preferred aspect of this embodiment, the cell or tissue culture is isolated from a non-human animal model whose genome comprises a homozygous disruption of one or more sestrin genes such that said genes do not produce functional sestrins, preferably sestrin 2 and a homozygous disruption of the ltbp4 gene such that said gene does not produce functional ltbp4.
In another preferred aspect of this embodiment, the cell or tissue culture is isolated from a non-human animal model whose genome comprises a heterozygous disruption of one or more sestrin genes such that said genes do produce only 50% or less of functional sestrins, preferably sestrin 2 and a homozygous disruption of the ltbp4 gene such that said gene does not produce functional ltbp4.
In another preferred aspect of this embodiment, the cell or tissue culture is isolated from a non-human animal model whose genome comprises a homozygous disruption of one or more sestrin genes, preferably sestrin 2 and a heterozygous disruption of ltbp4 such that said gene produces only 50% or less of functional ltbp4. Particularly preferred is that the cell or tissue culture is derived from lung or colon.
Embodiment (4) of the invention relates to a method for preparing the non-human animal model of embodiments (1) and (2), which comprises disrupting the sestrin and/or ltbp4 gene in a germ cell of a starting non-human animal. In a preferred aspect of this embodiment, the non-human animal and the starting non-human animal is a non-human mammal and the germ cell is an ES cell. The method may further comprise introducing the resulting ES cells into blastocysts, injecting the obtained blastocysts into respective non-human foster mothers and intercrossing the resulting chimeras.
Embodiment (5) of the invention is directed to a method for selecting an agent or agents for treating a symptom occurring in the animal model of the invention comprising: (i) applying one or more agents to be tested to the animal model of the invention; and (ii) determining whether one or more symptoms occurring in the animal model of the present invention have changed as a result of the application of said agent or agents. In a preferred embodiment the symptom is selected from a group consisting of pulmonary emphysema, cardiomyopathy and cancer. In a further aspect of the invention the agent, which is suitable for treating a symptom occurring in the animal model of the invention is a pharmaceutical. Furthermore, the present invention relates to the use of the agent, which is suitable for treating a symptom occurring in the animal model of the invention for the preparation of a pharmaceutical composition for the treatment of pulmonary emphysema. Moreover, the present invention is directed to a method of treatment of cancer and pulmonary emphysema using the agent, which is suitable for treating a symptom occurring in the animal model of the present invention.
Embodiment (6) of the invention is directed to a method selecting an agent that interferes with ROS production and TGF-β signaling comprising:
It is particularly preferred in said method that the cell or tissue culture is from lung or colon.
Embodiment (7) of the invention is a method to analyze whether pulmonary emphysema and/or cancer is caused by differential ltbp4 and sestrin gene or protein expression or expression level or by a defect in the ltbp4 and sestrin genes comprising the (i) characterization of the ltbp4 and sestrin gene or protein expression or expression level or of the ltbp4 and sestrin gene allele status of an individual having pulmonary emphysema and/or cancer, and the (ii) characterization of the ltbp4 and sestrin gene or protein expression or expression level or ltbp4 and sestrin gene allele status of a control individual. A difference in the ltbp4 and sestrin gene or protein expression or expression level or in the ltbp4 and sestrin gene allele status indicates that a defect in the ltbp4 and sestrin genes is involved in the pathogenesis of pulmonary emphysema and/or cancer.
Embodiment (8) of the invention relates to a method for diagnosing pulmonary emphysema or cancer comprising the (i) characterization the ltbp4 and sestrin gene or protein expression or expression level or the ltbp4 and sestrin gene allele status of an individual and (ii) characterization of the ltbp4 gene or protein expression or expression level or ltbp4 and sestrin gene allele status of a control individual. A difference in the ltbp4 and sestrin gene or protein expression or expression level or in the ltbp4 and sestrin gene allele status would indicate the presence of pulmonary emphysema and/or cancer in said individual. Among the determined expression levels it is the ltbp4 expression level that is an indicator of the severity of the pulmonary emphysema or of the cancer, while the sestrin expression level is a marker for the disease progression such that low expression levels are beneficial to the disease progression.
“Individual” used in connection with the embodiments (7) and (8) relates to an individual with suspected abnormal ltbp4 and sestrin gene allele status, i.e. a patient. “Control individual” refers to a healthy individual having normal ltbp4 and sestrin allele status.
In a preferred embodiment of the methods to analyze whether pulmonary emphysema and/or cancer are linked to ltbp4 and sestrin and for diagnosing pulmonary emphysema and cancer, the ltbp4 and sestrin gene allele status and the ltbp4 and sestrin expression or expression level are detected by genomic PCR, RT-PCR, Northern analysis, microarray (DNA chip) analysis or antibodies directed to the ltbp4 and sestrin proteins.
The methods of embodiments (5) to (8) are suitable to be performed in vivo and in vitro.
The animal model of the present invention can be used to dissect the molecular mechanisms controlling the sestrin/TGF-β pathway and for the identification and cloning of modifier genes able to modify, aggravate, reduce or inhibit the phenotype associated with pulmonary emphysema and cancer or other conditions occurring in the animal model of the invention. Moreover, the animal model can be used for identification of early diagnostic markers for cancer and/or pulmonary emphysema or other conditions occurring in the animal model of the invention. In addition, the animal model of the present invention can be used for the monitoring of the activity of agents useful in the prevention or treatment of cancer and/or pulmonary emphysema or other conditions occurring in the animal model of the invention and as a test model system for agents suspected of promoting or aggravating cancer and/or pulmonary emphysema or other conditions occurring in the animal model of the invention.
The invention furthermore explained in the following examples which are, however, not to be construed as a limitation of the invention.
Cell cultures: [129/SvPas] strain derived ES cells were grown on irradiated (32 Gy) MEF feeder layers in DMEM supplemented with 15% (v/v) preselected and heat inactivated fetal calf serum (FCS) (Linaris, Bettingen, Germany), 100 mM nonessential amino acids (Gibco), 0.1 mM mercaptoethanol (Sigma), 1000 U/ml of leukemia inhibitory factor (LIF) (Esgro®; Gibco/BRL), as described (De-Zolt S., et al. Nucleic Acids Res. 34:e25, 2006). Lung and colon fibroblast cultures from adult wild-type and sens−/− mice were established according to standard protocols as previously described (Koli K., et al. J Cell Biol. 167:123-33, 2004) and grown in Dulbeccos's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Life Technologies), 100 IU/ml penicillin and 50 μg/ml streptomycin. During the first two weeks in culture spontaneous immortalization occurred generating cell lines.
5′ RACE and sequencing: cDNAs from gene trap expressing ES cell lines were prepared from the polyadenylated RNA using a RoboAmp robotic device (MWG Biotech, Ebersberg, Germany) with a processing capacity of 96 samples/day. Samples of 2×105 cells were lysed in 1 ml of lysis buffer containing 100 mM Tris/HCl pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% LiDS and 5 mM DTT. Polyadenylated RNA was captured from the lysates by biotin-labeled oligo-d(T)-primers according to the manufacturers instructions (Roche Diagnostics Corp., Indianapolis, Ind., USA) and placed on streptavidin-coated 96-well plates (AB Gene, Surrey, UK). After washing, solid-phase cDNA synthesis was performed in-situ using random hexamers and Super-Script II RT (Invitrogen, Karlsruhe, Germany). To remove excess primers the cDNAs were filtered through Multiscreen PCR plates (Millipore Corp. Bedford, Mass., USA). The 5′ ends of the purified cDNAs were tailed with dCTPs using terminal transferase -TdT- (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions. For PCR amplification of the cellular sequences appended to the gene trap transcripts the following vector-specific primers were used: 5′-CTA CTA CTA CTA GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3′ (SEQ ID NO:4) and 5′-GCC AGG GTT TTC CCA GTC ACG A-3′ (SEQ ID NO:5); and for nested PCR: 5′-CTA CTA CTA CTA GGC CAC GCG TCG ACT AGT AC-3′ (SEQ ID NO:6) and 5′-TGT AAA ACG ACG GCC AGT GTG AAG GCT GTG CGA GGC CG-3′ (SEQ ID NO:7). Amplification products were directly sequenced using AB377 or ABI3700 sequencing machines (Applied Biosystems ABI, Foster City, USA).
ES cell injections, breeding and genotyping: WO77E06 (TBV-2; 129SvPas) ES cell derived chimeras were generated by injecting C57Bl/6 blastocysts. The resulting male chimeras were bred to C57Bl/6 females, and F1 agouti offspring containing the disrupted transgene were intercrossed to obtain homozygous F2 mice. Genotyping was performed on mouse tail DNA by genomic PCR using primers against the sequences flanking the gene trap insertion previously identified in ES cells by inverse PCR and sequencing.
Histology, histochemistry and immunohistochemistry: Paraffin sections of mouse tissues were prepared and stained using standard histology procedures. To visualize elastin and collagen fibers, microscopic slides were stained with Weigert's resorcin-fuchsin or Masson's trichrome stain as previously described (Sterner-Kock A., et al. Genes Dev. 16:2264-2273, 2002). Immunostainings using rabbit polyclonal anti- P-Smad2 antibodies were performed as described in Sterner-Kock et al (Ster ner-Kock A., et al. Genes Dev. 16:2264-2273, 2002)
Plastinations: Tracheas of lungs prepared from wild-type and mutant mice were injected at room temperature with 1-2 ml of E RTV silicone (Dow Corning, Midland, Mich., USA) using a 2 ml syringe. Curing Agent was added at a 1:10 ratio to the silicone polymer immediately prior to injection. Injection was stopped when the silicone was visible under the surface of both lungs. Following injection, the silicone was allowed to harden for 24-48 hours after which the specimens were placed in 10% potassium hydroxide solution for 5-7 days. and then left in boiling water for 8-12 hours to detach the tissue from the polymer. Following maceration in boiling water, the specimens were placed in 5% hydrogen peroxide for about 2 hours to complete the removal of residual tissues. Casts were then rinsed in running water overnight.
RNA isolation, RT-PCR and protein analysis: Total cellular RNA was isolated using RNeasy Mini kit (Qiagen) according to manufacturer's instructions. RNA concentrations and purities were determined spectrophotometrically (Ultrospec 3000, Amersham) as well as by agarose gel electrophoresis followed by ethidium bromide staining. RT-PCRs were performed according to standard protocols by using 75 ng of reverse-transcribed total RNA in a total volume of 50 μl. Real-time RT-PCR analysis of sestrin 2 gene expression in ES cells was performed using SYBR green chemistry (ABgene, Epsom, UK) and an iCycler (Biorad) machine. cDNA was synthesized from total RNA using random priming and Superscript II (Invitrogen) reverse transcriptase. PCR reactions were run as triplicates in 25-μl volumes on 96-well plates, with each reaction containing cDNA derived from 15 ng of total RNA, 1× ABsolute SYBR fluorescein mix (ABGene) and 5 pmol of each of the following primers: 5′-CCTGGAACGGAACCTCAAAATC-3′ (SEQ ID NO:8) and 5′-GGGCTTCAAGGAGCAGCAAG-3′(SEQ ID NO:9). Amplification reactions were allowed to proceed for 35 cycles at 94° C. for 15 s, 61° C. for 30 s, and 72° C. for 30 s. For Western blotting lysates of lung and colon fibroblasts were resolved by SDS-PAGE, transferred to nitrocellulose membranes and reacted with rabbit anti-sestrin 2 polyclonal antibody (PTG Inc., Chicago, Ill.).
Determination of intracellular peroxide levels: 3×105 lung or colon fibroblasts from wild type and sesn2−/− mice plated into 6 cm Petri dishes a day before were trypsinized, washed in serum free DMEM and resuspended in 5 ml of serum free DMEM. To some cultures H2O2 was added to a final concentration of 200 μM. After incubating for 3 hours dichlorodihydrofluorescein (DCF) was added to the cells at a final concentration of 30 μM and incubated for another hour. After that cell were washed in PBS, trypsinized and resuspended in 300 μl PBS which were subjected to FACS analysis.
The WO77E06 gene trap ES cell line was obtained by electroporating the pT1βgeo gene trap vector shown in SEQ ID NO:10 into TBV-2 (129SvPas) ES cells as previously described (Floss, T. & Wurst, W. Methods Mol Biol 185, 347-79, 2002). Gene trap expressing ES-cell clones were selected in 200 μg/ml G418 (Gibco/BRL), manually picked, expanded, and stored frozen in liquid nitrogen. The gene trap insertion in the 9th intron of the sestrin 2 (sesn2) gene was identified among the recovered clones by 5′ RACE as described in the Methods section and by sequence database analysis (Genbank at http://www.ncbi.nlm.nih.gov) using the BlastN algorithm. The gene trap insertion in the WO77E06 cell line was verified by genomic PCR (see Methods) using the following primers complementary to gene trap- and adjacent upstream intron sequences: 5′-CAGCCTTGAGCCTCTGGAGC-3′ (SEQ ID NO:11) and 5′-CTACCCTGAGAAGACGACCCG-3′ (SEQ ID NO:12). The verified WO77E06 ES cells were then converted into mice by blastocyst injection. F1 mice carrying the gene trap allele were intercrossed and the F2 offspring was mated to ltbp4−/− mice (WO 03/015505 A3) to obtain double knock-out mice.
Intercrossing F1 heterozygous offspring yielded homozygous offspring at a frequency consistent with a Mendelian inheritance pattern of the disrupted transgene, indicating that sesn2 is not required for development. Mutant mice developed normally after birth and were grossly indistinguishable from their wild type and heterozygous littermates.
The gene trap insertion in the 9th intron of the sesn2 gene induces a fusion transcript in which the exons upstream of the insertion are spliced in frame to βgeo. Because transcription is terminated at the gene trap's polyadenylation site, the processed fusion transcript encodes a truncated version of sesn2 and the βgeo reporter (
Wild type sesn2 transcripts were completely lost from lungs of homozygous WO77E06 mice whereas heterozygous lungs expressed about 50% of wild type levels (
129/Sv (D3) ES cells with a U3Cre gene trap vector (SEQ ID NO:16) insertion in the 5th intron of the ltbp4 gene were injected C57BL/6 blastocysts as previously described (WO 03/015505, Thorey, I. S. et al. Mol Cell Biol 18, 3081-3088, 1998). The resulting male chimeras were bred to C57BL/6 females and agouti offspring were tested for transgene transmission by tail blotting. Mouse tail DNA was cleaved with BglII which does not cut provirus. The DNA was fractionated on 1% agarose gels, blotted onto Hybond N nylon filters (Amersham/Pharmacia, Piscataway, N.J.) and hybridized to a 32P labeled provirus flanking sequence probe. Animals heterozygous for the gene trap insertion were backcrossed to C57BL/6 mice for at least six generations before analyzing the phenotypes in heterozygous and homozygous offspring. To obtain double mutant strains, heterozygous ltbp4+/− mice were crossed to homozygous sesn2−/− mice and the offspring was genotyped by tail DNA PCR as described in Example 1 using the following allele specific primers: ltbp4 wild type allele=5′-CCAATCTTGCTTCTTTGCTGAGC-3′ (SEQ ID NO:17) and 5′-GGCTCATGCTTGAATGTTTCAG-3′ (SEQ ID NO:18); ltbp4 gene trap (mutant allele)=5′-CCAATCTTGCTTCTTTGCTGAGC-3′ (SEQ ID NO:19) and 5′-ATCATGCAAGCTGGTGGCTG-3′ (SEQ ID NO:20); sesn2 wild type allele=5′-CTACCCTGAGAAGACGACCCG-3′ (SEQ ID NO:21) and 5′-GGACAAATCAAGGTTACACAGAAAAAAGTC-3′ (SEQ ID NO:22); sesn2 gene trap (mutant allele)=5′-CTACCCTGAGAAGACGACCCG-3′ (SEQ ID NO:23) and 5′-CAGCCTTGAGCCTCTGGAGC-3′ (SEQ ID NO:24).
To detect the extent of damage to the bronchioles, alveolar ducts and alveoli, we visualized the tracheobronchial trees of these animals by plastination. Plastination involves the instillation of silicone into the trachea of the isolated lung. Since the alveolar ducts prevent the silicone from entering the alveolar sacs, the technique provides an informative, three dimensional image of the tracheobronchial tree (Perry S. F., et al. Exp Lung Res. 26:27-39, 2000).
To test this, we visualized elastin and collagen in lung tissue sections using specific histochemical stains.
The increased collagen deposition in the lung and the increased P-smad2 levels in the colon of ltbp4−/−sesn2−/− mice suggested the TGF-β pathway might be similarly activated in the sesn2−/− mice despite their grossly normal phenotype. To test this we visualized P-smad2 in lung tissue sections as described above. In a preliminary experiment, we found that P-smad2 levels in sesn−/− lungs significantly exceeded the wild type levels, suggesting that loss of sestrin 2 activates TGF-fβ (
Recently published experiments in have shown that the inhibition of sesn2 expression by shRNA compromises the cell's ability to process ROS leading to ROS accumutation and oxidative stress (Budanov A. V., et al. Science. 304:596-600, 2004). In line with this, we expected antioxidant functions to be similarly compromised in cells derived from sesn2 knock out mice. To test this, we quantified basal and H2O2 induced ROS levels in MLFs using the dichlorodihydrofluorescein (DCF) fluorescence method.
SEQ ID NO:10 Gene trap vector pTβgeo
SEQ ID NO:16 Gene trap vector U3Cre
| Number | Date | Country | Kind |
|---|---|---|---|
| 06120714.8 | Sep 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP07/59227 | 9/4/2007 | WO | 00 | 3/13/2009 |
