However, patients with type 2 diabetes mellitus show a reduced VPC number and impaired functional activity. Previously, we demonstrated that p38 kinase inhibition prevents the negative effects of glucose on VPC number by increasing proliferation and differentiation towards the endothelial lineage in vitro. Moreover, the functional capacity of progenitor cells is reduced in a mouse model of metabolic syndrome including type 2 diabetes  and CD115 upon short term lentiviral delivery of ETS-specific shRNAs. Long term inhibition of ETS expression by lentiviral infection increased the number of cells with the endothelial markers CD144 and CD105.These data demonstrate that diabetes leads to dysregulated activation of ETS, which blocks the functional activity of progenitor cells and their commitment towards the endothelial cell lineage. Postnatal vessel formation, known as angiogenesis, was attributed to the migration and proliferation of preexisting mature endothelial cells db). Leprdb mice lack functional leptin receptors, become obese shortly after birth and are insulin resistant and hyperglycemic as adults The molecular mechanisms underlying the reduced endothelial progenitor cell number and function by high glucose are not yet clearly defined. Recently, Marchetti et al. demonstrated that high glucose levels (33 mM) increased apoptosis of endothelial progenitor cells in vitro and impaired the PI3-kinase/Akt pathway The aim of this study was to investigate potential signalling mechanisms in vasculogenic progenitor cells (VPC), which regulate the differentiation processes in vitro and in vivo.ETS (E26 transformation-specific DNA binding domain) is one of the largest families of transcriptional regulators that share a highly conserved DNA-binding domain (ETS domain). ETS transcription factors are downstream of the p38 MAP kinase, which has already been shown to be important for endothelial progenitor cell differentiation and proliferation We found that ETS DNA-binding activity is much higher in diabetic cells. Inhibition of the ETS activity leads to increase in impaired VPC number. Therefore this could be a potential mechanism to improve the impaired VPC number in patients with type 2 diabetes.Mononuclear cells (MNC) were isolated from fresh peripheral blood of healthy human volunteers or patients with type 2 diabetes.Written informed consent was obtained from all patients. The ethics review board of the Hospital of the Johann Wolfgang Goethe University of Frankfurt, Germany, approved the protocol, and the study was conducted in accordance with the Declaration of Helsinki.db mice) were obtained from Charles River Laboratories and used for the experiments at an average age of 12 weeks.Leptin-receptor-deficient mice  were plated on culture dishes coated with human fibronectin (Sigma) and maintained in endothelial basal medium  supplemented with 1 µg/ml hydrocortisone, 12 µg/ml bovine brain extract, 50 µg/ml gentamycin, 50 ng/ml amphotericin B, 10 ng/ml epidermal growth factor and 20% FCS. After 3 days of culture, more than 90% of the VPC express endothelial marker proteins KDR, VE-Cadherin and von Willebrand factor MNC were isolated by density gradient centrifugation with Biocoll separating solution  from fresh peripheral blood of healthy human volunteers or patients with type 2 diabetes as previously described Bone marrow of animals was harvested by flushing femurs and tibias with PBS. Mononuclear cells (BMC) were isolated by density gradient centrifugation using Ficoll . BMC were washed three times with PBS (800×g), counted and used for the experiments.In order to achieve long term gene silencing, DNA-oligonucleotides encoding shRNAs directed against ets1 or ets1 and ets2 were subcloned into pLKO.1-puro (Sigma). Lentiviral particles were produced in 293T packaging cells using a calcium phosphate co-precipitation method.The lentiviral particles were added (1∶5) to MNC immediately after isolation. A second round of infection was performed the next day and RNA was isolated on day 4. For long term experiments the number of adherent VPC was determined after staining with PE-conjugated antibodies against CD105 (BD Biosciences) and CD144 (BD Biosciences) at day 14 after infection. The biosafety level 2 experiments were approved by the “Bezirksregierung” Düsseldorf.www.genecards.org) these proteins are defined as follows: ETS: v-ets erythroblastosis virus E26 oncogene homolog (avian), ETS/PEA3: ets variant gene 4 (ETV4), EGR: early growth response. “ECL spots” are positive controls on the membrane and, according to the manufacturers specifications, can be used to normalize signal intensities. Therefore, after calculating the density of the spots using Tina software, the membranes were normalized to the average of their “ECL spot density” . Only binding sites were the densitometric analysis of duplicate spots and the corresponding dilutions yielded the same results were analyzed in more detail.After isolation of cells, nuclear and cytosolic fractions were separated using a commercially available kit according to the manufacturer's protocol (Pierce). Nuclear and cytosolic fractions were controlled for purity in Western blots with antibodies directed against the exclusively nuclear or cytosolic proteins Topoisomerase I (nuclear) and Hsp70 (cytosolic) as described previously Gene-specific mRNA expression was determined by real-time RT-PCR. RNA concentration was determined photometrically and aliquots of total RNA (2 µg) were used for cDNA-synthesis with the SuperscriptTM III First-Strand synthesis system for RT-PCR . PCR reactions were carried out on an Abi Prism 7000  using SYBRGreen PCR Master Mix . For relative expression comparison between different cells the ΔΔCT method was used, utilizing the 18S RNA as housekeeping gene. Primers were designed using the online-software Primer3. The following primer pairs were used:GCCGCTAGAGGTGAAATTCTTG, CATTCTTGGCAAATGCTTTCG18S RNA: CAACTGTGTGAGCCTGCTGT, GACAGGTCAGGGCTGATGATCD105: ATGGTCATGTGGCCAAGATT, GAGGATGCCATAGGACCAGACD115: GAGTCAACCCAGCCTATCCA, CCGAGGGGTAGTCATTCTCAETS1: TCTGGTGAACGTGAATCTGC, CGGAGGTGAGGTGTGAATTTETS2: TTGACAGCGACAAGAAGTGG, GCCATTCACGTCGTCCTTATMMP9: t test. Statistical significance was assumed at a value of p<0.05.Data are expressed as mean±SEM from at least 3 independent experiments unless stated otherwise. For in vitro data, statistical analysis was performed by a two-sided student db mice and compared them to their wild type littermates. Out of 450 transcription factors, the activity of ETS transcription factors was significantly increased in BMC of Leprdb mice compared to the wild type littermates (db mice (data not shown).Because diabetes leads to impairment in number and functional activity of progenitor cells in patients termates . Similardb mice, ETS transcription factors were activated under high glucose culturing conditions . DNA-protein binding arrays were performed and, similar as in Leprnditions . Having nditions . Indeed,nditions . Of noteTo demonstrate a causal link between ETS transcription factors and the reduced number of progenitor cells in type 2 diabetes, we transfected peripheral blood-derived mononuclear cells with control siRNA or siRNA against ETS1 and incubated them with high glucose for 3 days. High glucose levels induced a significant reduction of adherent VPC when transfected with control siRNA . In contIn the next set of experiments, we wanted to elucidate whether knock down of ETS transcription factors has an influence on the differentiation capacity of MNC. Therefore, we designed lentiviral shRNA vectors to permanently knock down ETS1 or ETS1 and ETS2 expression. Indeed, short term lentiviral knockdown of ETS1 expression resulted in a concomitant suppression of the known non-endothelial ETS-target genes MMP9 and CD115 The data of the present study demonstrate that increased nuclear DNA-binding activities of ETS1 and ETS2 transcription factors are crucial determinants of diabetes- and high glucose-induced reduced number of VPC. Moreover, increased DNA-binding activity of ETS-transcription factors inhibits the commitment of progenitor cells towards the endothelial cell lineage.Emerging evidence indicates that endothelial progenitor cells are involved in the maintenance of vascular homeostasis and their impairment may be conducive to vascular disease. Endothelial progenitor cell function is impaired in patients with coronary artery disease and diabetes The ETS protein family is a group of transcription factors that share a DNA-binding ETS domain and regulate the expression of a variety of genes including key ones involved in regulation of cell proliferation, differentiation and survival. Because of their critical role in basic cellular processes, dysregulation of ETS transcription factors can be found in many human diseases with the need for neovascularisation, such as cancer. ETS transcription factors have been implicated in the regulation of genes involved in homeostasis, vascular development and angiogenesis +KDR+ cells in the peripheral blood, has recently been shown as these cells predict the outcome in CAD-patients We could clearly show that ETS dysregulation by high levels of glucose leads to impairment of progenitor cell number as well as functional capacity. The importance of circulating endothelial progenitor cell number, measured as CD34Although the transcription factor ETS reduces VPC numbers, ETS activity is clearly important in adult angiogenesis Even though our findings demonstrate the importance of ETS1 in angiogenesis, ETS1 deficient mice develop normally except for an increased perinatal mortality Members of the ETS gene family are known to be expressed in the hematopoietic tissue and some of them play a pivotal role in hematopoietic cell development. The special importance of ETS1 regulation in differentiation of hematopoietic stem cells has previously been shown. During erythroid differentiation, ETS1 is downregulated and exported out of the nucleus. In contrast, during megakaryopoiesis, ETS1 increases and remains in the nucleus We clearly showed that glucose upregulates ETS DNA binding activity and thereby reduces VPC numbers. In addition, it has been shown that other pro-inflammatory stimuli/cardiovascular risk factors such as TNFα up-regulate ETS transcription factors Taken together, the present study demonstrates a critical role for ETS transcription factors on VPC number and function. In vitro and in vivo high glucose levels increased ETS DNA-binding and thus most likely transcriptional activity. Inhibition of ETS1 and ETS2 expression counteracts the reduction of VPC number by enhancing endothelial lineage commitment. Given the fact that ETS transcription factors regulate a plethora of genes


The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues. Every year over 50,000 people die from rabies worldwide, primarily due to the poor availability of rabies vaccine in developing countries. However, even when vaccines are available, human deaths from rabies occur if exposure to the causative virus is not recognized and vaccination is not sought in time. This is because rabies virus immunity induced by the natural infection or current vaccines is generally not effective at removing disease-causing rabies virus from brain tissues. Our studies provide insight into why this is the case and how vaccination can be changed so that the immune response can clear the virus from brain tissues. We show that the type of immune response induced by a live-attenuated rabies virus vaccine may be the key. In animal models, live-attenuated rabies virus vaccines are effective at delivering the immune cells capable of clearing the virus into CNS tissues and promote recovery from a rabies virus infection that has spread to the brain while conventional vaccines based on killed rabies virus do not. The production of rabies-specific antibody by B cells that invade the CNS tissues is important for complete elimination of the virus. We hypothesize that similar mechanisms may promote rabies virus clearance from individuals who are diagnosed after the virus has reached, but not extensively spread, through the CNS. Rabies viruses spread from peripheral sites of entry to the central nervous system (CNS) tissues via axonal transport thereby bypassing the specialized features of the neurovasculature known as the blood-brain barrier (BBB). Once the virus reaches CNS tissues three alternative outcomes are likely: (1) the BBB remains intact and the infection is lethal due to the absence of an antiviral CNS immune response (2) immune effectors cross the BBB and mediate a CNS antiviral immune response with extensive immunopathology that contributes to the disease, or (3) immune effectors cross the BBB and clear the virus from the CNS without significant pathological consequences. It is well known that in humans naturally infected with rabies virus the latter outcome is exceedingly rare. In addition, CNS inflammation is generally limited in individuals who succumb to rabies Unlike humans where rabies viruses may take weeks to reach the CNS from the site of exposure −/− mice on a C57BL6 background were obtained from the in-house breeding colony at Thomas Jefferson University. RAG-2−/− mice on a 129/SvEv background were obtained from Taconic . Mice were infected or immunized intranasally (i.n.) with 105 focus forming units of CVS-F3, CVS-N2c or UV-inactivated CVS-F3 in PBS as previously described −/− mice were treated intraperitoneally with 1 mg of the monoclonal, rabies virus glycoprotein-specific, virus-neutralizing antibody 1112 in 500 µl of saline or with the vehicle alone at the time points noted in the figure legends. All procedures were carried out according to the protocols approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University.Eight to 12-week old wild-type control 129/SvEv and C57BL6 mice and JHD™II fluorimeter. The amount of Na-fluorescein in the CNS tissue is normalized to its level in serum by (µg of Na-fluorescein in CNS tissue/mg of tissue)/(µg of Na-fluorescein in serum/µl of serum) and is expressed as a fold increase in fluorescence uptake by comparison with the results obtained from naïve controls.BBB integrity was assessed by quantifying the leakage of a low molecular weight fluorescent marker  from the circulation into CNS tissues as previously described For immunohistochemical analysis, brains from perfused mice were snap frozen in Tissue-Tek O.C.T. Compound , sectioned using a Thermo Shandon cryostat , and fixed in either 80% acetone or 95% ethanol. Immunoglobulin (Ig) was detected using either biotinylated monoclonal rat anti-mouse kappa light chain (1 hour at 1∶50)  followed by Alexa Fluor 568 streptavidin (1 hour at 1∶1000)  or the VECTASTAIN ABC-AP KIT with polyclonal rabbit anti-mouse biotinylated IgG (1∶200) developed using the peroxidase antiperoxidase method and 3′3-diaminobenzidine as substrate  according to the manufacturer's protocol. For the additional staining shown in one of the figure panels (#3D), a 1 hour incubation with 1 mg/ml 1112 was performed prior to detection of Ig. To assess virus infection sections were stained for 1 hour with FITC-conjugated anti-rabies virus nucleoprotein monoclonal antibody (1∶50) . Photographs were taken with a Nikon digital camera on an Olympus BX-60 microscope.Total RNA was isolated from CNS tissue samples and mRNA expression levels of rabies virus nucleoprotein, CD4, CD8, IFN-γ and L13 in CNS tissues were measured by quantitative reverse-transcriptase (RT)-PCR as previously described 2) and incubated with anti-CD16/32 (1 ug/106 cells)  antibody to prevent non-specific binding. Cells were washed in PBS and incubated with anti-mouse CD19 (1∶1000)  and MHC class-II (1∶1000)  antibodies. Phenotypic characterization of antibody-labeled cells was performed on a BD-FacsCaliber Flowcytometer. CD19-MHC class II double-positive cells were defined as B cells. Numbers of rabies virus-antigen specific antibody secreting B cells were assessed using Millipore Multiscreen HA® ELISPOT plates coated with 5 ug/mL of UV-inactivated whole rabies virus. Peripheral blood or brain derived mononuclear cells were suspended in RPMI media supplemented with 25 mM HEPES and 10% FBS and 200,000 cells were incubated in each well for 18 hours. Plates were washed and bound rabies virus-specific antibodies were detected by addition of alkaline-phosphatase conjugated anti-mouse IgG antibody (1∶500)  followed by BCIP/NBT substrate. Spots were counted using a dissecting microscope.Mononuclear cells were prepared from peripheral blood collected by retro-orbital bleeding in heparinized capillary tubes by centrifugation at 300 g for 20 minutes. The white cell layer was washed in PBS twice before analysis. Mononuclear cells were isolated from CNS tissues as described elsewhere by isolation at the interface of a 30/70 Percoll (Sigma) step gradient centrifuged at 800 g for 25 minutes Results are expressed as the mean±standard error mean (S.E.M.). Statistical significance of the differences between groups was tested using the Mann-Whitney test and the symbol * indicates a p value<0.05.Mouse models are not considered to be particularly suited to studies of post-exposure prophylaxis (PEP) with rabies due to the rapid spread of the viruses to the CNS. However, our prior studies suggest that the lethal outcome of rabies in mice is more a consequence of the inability to deliver immune effectors into CNS tissues than its spread −/− mice lack B cells but have functional T cells and, unlike RAG-2−/− mice, which lack both T and B cells, are capable of elevating fluid-phase BBB permeability in response to the infection . This raises the possibility that T cell activities may be able to partly control the virus infection independently of antibody. To gain insight into the contributing T cell subsets, we compared the levels of CD4, CD8 and IFN-γ mRNAs in CNS tissues from wild-type conventional mice 24 days after CVS-F3 infection when there is little virus replication remaining (see below) and JHD−/− mice 40 days after infection. As can be seen in −/− mice but IFN-γ mRNA levels have remained relatively high and may therefore be contributing to the control of virus replication.JHDnfection . These mogresses . Howeverght gain . At the −/− mice . These J−/− mice to clear CVS-F3 from the CNS reaffirms the importance of rabies virus-specific antibodies in this process. However, little is known with respect to how these antibodies may be delivered to infected CNS tissues. Our studies of mice clearing CVS-F3 suggest that the leakage of naturally developing antibodies from the circulation into the CNS tissues may be minimal since elevated BBB permeability occurs before serum antibody titers peak −/− mice were treated with 1 mg of the mouse IgG1 monoclonal rabies virus-neutralizing, glycoprotein-specific antibody 1112, which is highly effective in post-exposure treatment models −/− mice  and after BBB permeability has peaked y higher . This suPrompt administration of PEP is the recommended course for an individual who has come in contact with a rabid animal. Since this prevents the development of clinical rabies it is impossible to be certain how many of the tens of thousands of people who receive PEP on an annual basis have actually been infected with the virus. It is also impossible to know how far the rabies virus may have spread before being cleared by the immune effectors provided or induced by PEP and the infection. The commonly held view that pathogenic wildlife rabies virus that has spread to the CNS cannot be cleared by immune mechanisms is supported by the absence of significant immune cell infiltration into the CNS tissues of individuals who die from rabies −/− mice, which lack B cells but have functional CD4 and CD8 T cells, often survive CVS-F3 infection over extended periods despite being unable to clear virus from CNS tissues and exhibiting neurological symptoms. This leads us to conclude that elements of the T cell response, likely including IFN-γ production by CD4 and CD8 T cells, can control certain features of the infection that make significant contributions to its lethality but that antibody is required for virus clearance. To examine the contribution of circulating antibody to virus clearance from CNS tissues, we administered high levels of the rabies virus neutralizing mouse monoclonal 1112 antibody to CVS-F3-infected JHD−/− mice during the stage of infection when BBB permeability is maximal. While leakage of a 150 kDa molecular weight marker from the circulation into the CNS tissues of CVS-F3-infected mice is minimal over a 4-hour period in vivo appears to be relatively low as considerably greater amounts of the antibody can bind to the cells when applied to tissue sections in vitro. While it is possible that even low levels of virus-neutralizing antibody may impact the replication and spread of the virus while BBB permeability is enhanced, treatment of CVS-F3-infected JHD−/− mice with 1112 antibody failed to clear the virus.To gain further insight into the mechanism of rabies virus clearance from the CNS tissues, we have used gene-deleted mice to study the role(s) of different antiviral immune effectors in the CNS tissues of mice clearing the attenuated rabies virus CVS-F3. Mice lacking T and B cells cannot clear this virus and die from the infection in vitro. This leads us to conclude that the high levels of antibodies required for rabies virus clearance from the CNS tissues are produced at the site of infection rather than diffusing in from the circulation. In this case, passively administered antibody during PEP would primarily impact virus in the periphery and an active immune response leading to elevated BBB permeability and immune effector delivery to the CNS tissues would likely be required to clear virus from the CNS.It should be noted with respect to the origin of the antibodies that participate in rabies virus clearance that serum rabies virus-specific antibody titers peak some time after BBB integrity has been restored As certain of the aspects of BBB function required for immune cell infiltration are unchanged by CNS infection with pathogenic rabies viruses


WWOX, the gene that spans the second most common human chromosomal fragile site, FRA16D, is inactivated in multiple human cancers and behaves as a suppressor of tumor growth. Since we are interested in understanding WWOX function in both normal and cancer tissues we generated mice harboring a conditional Wwox allele by flanking Exon 1 of the Wwox gene with LoxP sites. Wwox knockout (KO) mice were developed by breeding with transgenic mice carrying the Cre-recombinase gene under the control of the adenovirus EIIA promoter. We found that Wwox KO mice suffered from severe metabolic defect(s) resulting in growth retardation and all mice died by 3 wk of age. All Wwox KO mice displayed significant hypocapnia suggesting a state of metabolic acidosis. This finding and the known high expression of Wwox in kidney tubules suggest a role for Wwox in acid/base balance. Importantly, Wwox KO mice displayed histopathological and hematological signs of impaired hematopoeisis, leukopenia, and splenic atrophy. Impaired hematopoeisis can also be a contributing factor to metabolic acidosis and death. Hypoglycemia and hypocalcemia was also observed affecting the KO mice. In addition, bone metabolic defects were evident in Wwox KO mice. Bones were smaller and thinner having reduced bone volume as a consequence of a defect in mineralization. No evidence of spontaneous neoplasia was observed in Wwox KO mice. We have generated a new mouse model to inactivate the Wwox tumor suppressor gene conditionally. This will greatly facilitate the functional analysis of Wwox in adult mice and will allow investigating neoplastic transformation in specific target tissues. WWOX) was cloned and identified as a potential tumor suppressor gene mapping to the chromosome region 16q23 WWOX gene spans >1.1 Mb and overlaps the common chromosomal fragile site, FRA16D, the second most common site for chromosomal breakage, instability and rearrangement of the whole genome WWOX locus have been described in various human cancers WWOX expression in multiple human neoplasias including breast, prostate, ovarian, lung and liver cancer WW domain-containing oxidoreductase  WWOX as a tumor suppressor is supported by in vitro studies with human cancer cells and in vivo studies using mouse models. Several studies reported that ectopic WWOX expression suppressed the in vivo tumorigenicity of various human tumor types including, breast Wwox had increased spontaneous tumor incidence. Using a conventional mouse knockout approach Aqeilan et al. −/−Wwox mice by the age of 2.5 weeks and as early as postnatal day 3. All Wwox null mice died by 3–4 wk of age precluding any further studies of adult animals. However, development of spontaneous lung papillary carcinoma was observed in adult +/−Wwox mice. Aqelian et al +/−Wwox mice treated with chemical carcinogens displayed an increased incidence of lymphomas and lung tumors when compared with wild type littermates. Using a gene trap approach we developed a Wwox hypomorphic mouse model Wwox hypomorphs had an increased incidence of spontaneously arising B-cell lymphomas. In addition, several female developed multiple neoplasias. Together, the in vitro and in vivo studies have provided significant evidence for Wwox as a tumor suppressor.The role of Wwox plays in the multiple tissues it is expressed. As mentioned, Wwox KO mice generated using conventional techniques die by four weeks of age thus impeding any studies in adult tissues. Therefore, we report here the generation of mice with a conditional allele for Wwox ablation for better understanding the roles of Wwox in different tissues and in pathological conditions.We are interested in understanding in more detail the normal and tumor suppressive role Wwox gene expression using the Cre-Lox site-specific recombination system. Our approach, illustrated in Wwox genomic sequences by flanking Wwox Exon 1 with two LoxP-recombination sites to generate a “floxed”-Wwox allele. We successfully targeted mouse ES cells and generated chimeric founders having germline transmission and subsequently bred a floxWwox mouse.We devised a strategy for conditional ablation of pgk-neo plasmid could impair Wwox gene transcription we determined whether the floxed-Wwox allele had any effect on viability and fertility. Breeding of heterozygous (+/floxWwox) mice from two different founders resulted in pups having genotypes with the expected Mendelian ratios demonstrating that the floxed allele had no effect on embryonic development or viability. Mating of flox/floxWwox males with flox/floxWwox females resulted in normal litter size, genotype frequencies and pups had normal postnatal development. These results showed that the “floxed”-Wwox allele containing the insertion of the ∼2 Kb neomycin resistance gene in the 5′ transcriptional control region of the Wwox gene had no detrimental affect on Wwox functions required for normal mouse growth, survival or reproduction. Therefore, this mouse model will be a useful tool for analyzing Wwox function(s) in specific mouse tissues.Since it was possible that the inserted Wwox allele resulting in the insertion of the two LoxP-recombination sequences flanking Exon 1 however it is possible that small changes to the inserted sequence could have been introduced during the procedure. Therefore, to determine whether Cre-recombinase could utilize the inserted LoxP sites to delete Wwox Exon 1, we generated mice combining the floxWwox allele with the Cre-recombinase gene under the control of the adenovirus EIIA promoter. The adenovirus EIIA promoter directs transcription of Cre-recombinase at the zygote stage of embryonic development resulting in deletion of floxed sequences in all cells of the developing embryo including the germ cells Cre-recombination was successful we analyzed genomic DNA using PCR with oligonucleotide primers specific for wild-type or Cre-deleted Wwox sequences  of pups having one Wwox allele with wild-type sequences and one Wwox allele with Exon 1 deleted (+/ΔCreWwox). Since the ΔCreWwox allele was present in the germ cells a F1×F1 cross should result in pups having all possible genotypes  with Mendelian ratios of 1∶2∶1 (+/ΔCre mice were fertile having litter sizes ranging between 3 to 11 pups (average  = 7.75). Three-day old pups were genotyped by PCR (2-test). To demonstrate that our targeting strategy lead to complete ablation of Wwox expression we performed immunoblot analysis of total protein extracts from a variety of tissues . Although some Wwox KO mice can survive up to 3 weeks of age, the mortality rate was very high. As early as 72 h after birth 43% (15 of 35) of Wwox KOs had died and 77% had died by 17 days after birth and no mice survived past weaning. These findings demonstrated that ablation of Wwox expression by deleting Exon 1 using our Cre-loxP strategy lead to growth retardation and postnatal death in agreement with the observations of Aqeilan et al Ablation of at birth . To furtWwox KO mice we performed necropsies and analyzed blood chemistries of mice at postnatal day 18. Necropsy results found significantly reduced relative weights of multiple organs and increased brain weight in KO mice consistent with wasting   . The hisWe also compared the histology of thymus that also displayed reduced relative weight (p = 0.05). We observed that in spite of preserving relatively normal organ architecture, the cortex of the thymus of KO mice is significantly thinner than that of wild type counterparts .3 cells per ul  while Wwox KO mice showed signs of leukopenia with lower WBC at an average of 4.2×103 cells per ul . No major differences were observed in the relative representation of the various white blood cell subtypes (not shown). However, one of the two KO mice analyzed displayed 3% of nucleated red blood cells (NRBC)/100 WBCs, a clear sign of severe anemia, while none of the WT mice showed any NRBCs. In summary, the histopathological and hematological findings suggest significantly impaired hematopoiesis in Wwox KO mice affecting white and red blood cell lineages.Given these histopathological findings we proceeded to perform some hematological analyses. White blood counts (WBC) were estimated manually on blood smears obtained from mice of the different genotypes. WBC for wild-type mice was estimated at an average of 9.45×10Wwox KOs compared with wild-type and heterozygotes  . In addition, KO mice displayed dramatically reduced bicarbonate levels (CO2) in blood (p = 0.006) indicative of serious disturbance in the acid/base balance compatible with metabolic acidosis.Most of the parameters measured by blood chemistry were not significantly different in the ozygotes . HoweverWwox KO mice had defects in bone formation Wwox KO mouse. We used micro-computed tomography (micro-cT) and bone histomorphometry to measure several parameters of endochondral bone formation. Femurs of 18-day old mice were used for these analyses. Micro-cT analyses were used to obtain high-resolution scans and constructed 3-D images to examine morphological features  of the tissue. The bone volumes of femurs from KO mice were reduced >66% in comparison to femurs from WT and HET mice  in femurs of KO mice (p = 0.02). This indicates that osteoblasts of Wwox KO have a defect in mineralization resulting in reduced bone formation.Therefore, to understand the defect of bone formation in Wwox KO mice were subjected to complete necropsies, X ray analyses and multiple organ histopathology. We did not observed any lesions compatible with neoplastic growth by any of the methodologies. Importantly, X ray analyses did not reveal any bone lesions compatible with osteosarcoma.A total of nine 18d old WWOX tumor suppressor gene is inactivated in many types of human cancers by genomic rearrangements, aberrant mRNA splicing, homozygous deletions and epigenetic silencing Wwox KO mice do not survive to adulthood previous studies of Wwox tumor suppression required chemical carcinogen treatment of +/−Wwox mice Wwox tumor suppressor gene.The Wwox allele. We utilized adenovirus EIIA-regulated Cre-recombinase transgenic mice to delete Wwox Exon 1 in all mouse tissues and ultimately generated Wwox KO mice. We found that Wwox KO mice suffered from severe metabolic defect(s) resulting in significant growth retardation, hypoglycemia, impaired hematopoeisis, and signs of metabolic acidosis that ultimately lead to premature death of all mice by 3 weeks of age. Bone formation of Wwox KO was also significantly affected. Bones from KO mice were analyzed and found to be smaller and thinner having reduced bone volume as a consequence of a defect in mineralization.In this report we describe the generation and initial characterization of a novel mouse model with a conditional Wwox KO mice had severe metabolic defects. Hypoglycemia can result from multiple causes such as disorders in carbohydrate, fat or amino acid metabolism as well as endocrine system disorders. Since Wwox is expressed in a variety of endocrine, neuroendocrine and non-endocrine tissues it is not possible from these studies to identify the cause of hypoglycemia. The combination of increased BUN with reduced bicarbonate levels (hypocapnia) suggests impaired kidney function and a disturbance in the blood acid-base balance that would result in acidosis. Protracted acidosis disturbs many bodily functions and can lead to growth retardation, bone disease, possibly total kidney failure and ultimately death. Renal tubular acidosis (RTA) is a pathology that occurs when the kidneys fail to excrete acids into the urine, which causes the blood to remain too acidic due to defects of cells in the proximal and distal convoluted tubules. The convoluted tubules of healthy kidneys help maintain acid-base balance by excreting acids into the urine and returning bicarbonate to the blood. Interestingly, we have previously demonstrated that Wwox is highly expressed in distal convoluted tubules and to a lesser extent in the proximal tubules of the kidney in mice  that ultimately leads to death. In addition, as significant contributing factors to morbidity and death, we also observed severe histopathological abnormalities affecting hematopoietic organs such as spleen and thymus in addition to white blood cell counts compatible with a significant suppression on hematopoiesis affecting the Wwox KO mice. Specifically, the spleens of Wwox KO mice displayed signs of atrophy with dramatic reduction in red pulp cellularity. All studies indicate that Wwox KO mice suffered from anemia and leukopenia. Anemia can be also intimately linked to kidney function and to metabolic acidosis. Additional studies will be required to determine the exact causes of the impaired hematopoiesis observed as a result of Wwox ablation.The results of the blood chemistry analysis demonstrated that  in mice  and humaWwox KO mice have been described to suffer alterations in bone metabolism Wwox that contribute to the variety of abnormalities we and others have observed. Therefore, we wanted to determine whether our mouse model of EIIA-Cre mediated ablation of Wwox expression recapitulated the observed defects in normal mouse bone development. We used micro-cT and histomorphometry to measure several static bone parameters. We observed a decrease of bone formation in Wwox KO mice associated with a reduction of mineralization and concomitant increase in osteoid compared to WT and HET mice. Proper bone formation results from a fine balance between bone formation and bone resorption. This balance is mostly maintained by the activity of two bone cell types, osteoblasts and osteoclasts, that make bone and degrade bone respectively. We measured osteoblast activity and found no significant differences in the number of active and non-active osteoblasts in femurs between genotypes. Measurements of osteoclast activity did not show any differences in femurs from mice of any genotype either. This is in sharp contrast to the observations of Aqeilan et al Wwox KO mice have reduced bone formation due to a bone mineralization defect. This is consistent with previous results that demonstrated a significant defect in differentiation of Wwox KO calvarial osteoblasts ex vivo compared to WT osteoblasts Wwox allele is sufficient for full function.Wwox KO mice developed focal lesions along the femoral diaphysis compatible with periostal osteosarcoma, lesions observed in one mice as early as 3d of age Wwox KO mice analyzed macroscopically, histopathologically and radiographically. Furthermore, none of the long bones that were subjected to MicroCT analyses revealed any malignant lesions either. The reason(s) for the discrepancies between studies remain to be determined.Importantly, Aqeilan et al. also reported that 4 of 13 (31%) mice Wwox gene was identified in rats that showed severe growth retardation, experienced epileptic seizures and died without reaching maturity lde for lethal dwarfism with epilepsy, was identified as a 13-bp deletion in Exon 9 of the Wwox gene resulting in a frame shift mutation causing aberrant amino acid sequences (371–424aa) at the C-terminal of the Wwox protein. Remarkably, lde/lde rats displayed phenotypic characteristics similar to Wwox KO mice including severe dwarfism and early lethality. The cause of dwarfism is not clear, however, lde/lde rats had slightly lower bone density and no osteosarcomas developed during their short life span lde/lde rats displayed significantly higher levels of BUN compared to normal rats Wwox KO mice.Very recently a spontaneous mutation in the Wwox locus. We demonstrated that homozygous Cre-recombinase mediated deletion of Wwox Exon 1 lead to complete ablation of Wwox expression. Loss of Wwox expression by this approach resulted in a phenotype similar to the previously reported Wwox KO generated by conventional techniques making this floxWwox strain an important reagent for studying Wwox function in normal mouse physiology and tissue specific tumorigenesis.In conclusion, we have generated a novel mouse model with a conditional allele at the Mice used in this were kept in a clean, modified-barrier animal facility, fed regular commercial mouse diet  under controlled light (12L∶12D) and temperature (68–74°F). All animal research was conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) at the University of Texas, M.D. Anderson Cancer Center, Science Park Research Division, following international guidelines and all research was approved by the corresponding Institutional bioethics committee (IACUC).Wwox targeting construct was generated using ∼6 Kb of Wwox gene obtained from BAC clone 9N99 containing a 5′ portion of the Wwox gene from 129/svJ mouse genomic DNA. Primers used for generating the targeting construct were: Right arm forward, 5′-CAAAACCCGGAAACTGGATTACAGC-3′;The 5′-CCTAGACTGTGCTGCTCCAA-3′; Left arm forward, 5′-GAGGAGGAGGAGGAGGGAAGTAGAGG-3′; Left arm reverse, 5′-CAGCTACTGGCGGGAAAGGG-3′. First, a ∼4 Kb genomic sequence containing Wwox Exon1 and a portion of Intron 1 was PCR amplified and cloned into plasmid pK-11 neomycin transferase gene under control of the phosphoglycerol kinase promoter (pgk-neo) used as the positive-selection element and a single LoxP Cre-recombinase recognition site. Another LoxP Cre-recombinase recognition site was inserted into Wwox Intron 1 in the same orientation as the pK-11 LoxP site in order to “flox” Wwox Exon 1 (floxWwox) resulting in the plasmid pK-11-Wwox-flox. The left arm of the targeting construct was generated by cloning ∼2 Kb of the Wwox 5′ upstream sequence into the plasmid pBJ101-DT diphteria toxin A-chain gene (DT-A) used as the negative-selection element. The final targeting construct was generated by transferring the DT-A gene and Wwox left arm sequences to pK-11-Wwox-flox plasmid  for generation of targeted ES cells and mouse chimeras. ES cell clones were electroporated with the Wwox targeting vector that had been linearized by digestion with Not I restriction endonuclease. Transfected ES cells were selected for G418-resistance and individual clones screened by Southern hybridization. Genomic DNA was digested with Bgl II and hybridized to a probe corresponding to a region of Wwox Intron 1 outside of the targeting construct sequences  containing protease inhibitors and incubation at 4°C for 30 min. with constant agitation. Insoluble material was then removed by centrifugation at 10,000 x g for 10 min. at 4°C. Protein concentration was determined using the BCA method . Western blot analysis was performed as previously described 2 asphyxiation, organs harvested and immediately weighed using a Mettler AE50 analytical balance.Organs were then fixed in formalin for 24 hours, then 70% ethanol and embedded in paraffin. Tissue sections were stained in H&E or processed for immunostaining. Blood was collected immediately following euthanasia by intracardiac puncture on the left ventricule and allowed to clot at room temperature for 30 minutes. Serum was collected following centrifugation for 10 minutes at 14,000 rpm at 4°C. Blood chemistries were measured using serum from each individual mouse using the Olympus AU400 automated chemistry analyzer. Manual differential and WBC were obtained from feathered blood smears using fresh whole blood from each individual animal.Mice were euthanized by COLimbs were dissected and soft tissue was carefully removed then fixed in formalin for 48 hours followed by 70% ethanol. Microcomputed tomography (micro-cT) was performed at the Baylor College of Medicine MicroCT Core facility. Bone histomorphometric analyses were performed at the UT MD Anderson Bone Histomorphometry Core Laboratory  following the guidelines established by the American Society for Bone and Mineral Research (ASMBR) histomophometry nomenclature committee.


Wolbachia are obligate endosymbiotic bacteria maternally transmitted through the egg cytoplasm that are responsible for several reproductive disorders in their insect hosts, such as cytoplasmic incompatibility (CI) in infected mosquitoes. Species in the Culex pipiens complex display an unusually high number of Wolbachia-induced crossing types, and based on present data, only the wPip strain is present.wPip strain of Wolbachia revealed the presence of 60 ankyrin repeat domain (ANK) encoding genes and expression studies of these genes were carried out in adult mosquitoes. One of these ANK genes, pk2, is shown to be part of an operon of three prophage-associated genes with sex-specific expression, and is present in two identical copies in the genome. Another homolog of pk2 is also present that is differentially expressed in different Cx. pipiens group strains. A further two ANK genes showed sex-specific regulation in wPip-infected Cx. pipiens group adults.The sequencing of the wPip suggest an important role in Wolbachia biology, and the gene family provides both markers and promising candidates for the study of reproductive manipulation.The high number, variability and differential expression of ANK genes in  Wolbachia are obligate endosymbiotic bacteria that are maternally transmitted through the egg cytoplasm and are responsible for several reproductive disorders in arthropods, such as cytoplasmic incompatibility (CI) in infected Culex mosquitoes [Wolbachia are not found in mature sperm, they can modify developing sperm, possibly via chromatin binding proteins [Wolbachia-infected females therefore produce a higher mean number of offspring than uninfected females. This unidirectional CI enables Wolbachia to rapidly invade uninfected populations [Wolbachia.squitoes ,2 and maproteins , such thulations , and proulations ,6. BidirwMel strain [Wolbachia strain found in Drosophila melanogaster, revealed an unusually high number of ankyrin repeat domain (ANK) encoding genes. Ankyrin repeats, consisting of around 33 residues, have been identified in a large number of proteins [Ehrlichia phagocytophila was detected in the host cytoplasm and found to be associated with chromatin suggesting a possible role in host cell gene expression [Nasonia wasps, the control of host cell cycle timing at karyogamy appears to be disrupted in CI and inhibition of CDK1 has been proposed as a possible mechanism [Wolbachia-induced CI [The genome sequence of the l strain , a CI-inproteins . Ankyrinproteins . Althougpression . ANK proechanism ,12. Takeduced CI .Cx. pipiens complex display an extremely high number of Wolbachia-induced crossing types between populations, with a high frequency of uni- or bidirectional incompatibilities [wPip strain of Wolbachia, responsible for CI in Cx. pipiens mosquitoes, has been found in the nucleotide sequences of ftsZ [wsp (surface protein)gene [wPip genome was undertaken partly in order to attempt to resolve this discrepancy. Interestingly, sequence analysis of some ANK genes found in wPip revealed variation in both nucleotide sequence and predicted amino acid sequence for two prophage associated ANK genes, pk1 and pk2, between wPip-infected Cx. pipiens colonies [wAu strain of Wolbachia, found in Drosophila simulans, is closely related to the wMel strain but does not normally induce CI [pk2 in wAu contains a premature stop codon not present in the wMel homolog, which suggests it could be a candidate gene for involvement in CI in Drosophila [Species in the bilities -15. Desp of ftsZ  or in thein)gene . Sequenccolonies . The wAunduce CI . The homosophila .Cx. pipiens was detected for the pk2 gene, a characteristic that might be expected for genes involved in the specific modification and rescue functions between incompatible strains. Any differential expression of ANK genes between male and female wPip infected adult Cx. pipiens mosquitoes would suggest an important function of these genes in the interaction between Wolbachia and its insect host. How Wolbachia differentially modify sperm in males as well as rescue in females is as yet unknown, but could potentially involve variability in the expression and activity of Wolbachia genes in male and female insect hosts. Variable gene expression in Wolbachia is not thought to occur at a high rate, as only a small number of regulatory genes have been identified in the Wolbachia genomes sequenced to date [wPip in Cx. pipiens adult mosquitoes.Variable expression between sexes and strains of  to date . In thiswPip genome revealed 60 ANK genes, which are numbered sequentially in Table wPip ANK genes are contained in several chromosomally integrated prophage regions, similar in sequence to the wMel WO-B prophage region [pk1 and pk2, homologues of the wMel genes WD0596 and WD0636 respectively and previously shown to vary between incompatible Culex strains [wPip_ANK8, wPip_ANK14 and wPip_ANK56 in the case of pk1 and wPip_ANK12 and wPip_ANK25 in the case of pk2. Two sequence variants of pk2 in wPip from different Cx. pipiens group colonies have been previously described and were named a and b. The wPip_ANK16 gene is also homologous to the pk2 genes/WD0636 in the wMel strain and is present in wPip in all the infected Cx. pipiens group colonies listed. A third pair of identical prophage-associated genes are also present, wPip_ANK13 and wPip_ANK26, which are homologues of WD0637. Thus in total there are 56 unique ANK genes present in the wPip genome.Analysis of the e region . The ANK strains , are herwMel ANK genes have clear homologues in the wPip genome, which might reflect the high degree of heterogeneity in this group of genes. Thus, when likely paralogous groups  and identical copies are taken into account, 37 of the 60 identified wPip ANK genes in the wPip genome do not have any clear homologues in the wMel genome. By way of comparison, the wBm strain of Wolbachia, thought to be a nutritional mutualist in the filarial nematode Brugia malayi, encodes only five ANK proteins [wPip ANK encoding genes.Only 15 of the 23 proteins , three owPip_ANK57 gene showed very low expression in Pel female extracts and no detectable expression in Pel male RNA extracts. wPip_ANK2 and wPip_ANK49 showed low levels of expression in both Pel male and female RNA extracts. RT-PCR analysis also suggested that wPip_ANK38 is highly expressed in both sexes.Transcripts were detected for all of the ANK encoding genes. For the majority, expression in adult males and females of the Pel colony was not obviously different based on agarose gel electrophoresis of RT-PCR products. The wPip_ANK12 and wPip_ANK25, previously together named pk2 [Cx. pipiens colony from Sri Lanka (Sumo Cyppe). Quantification of expression by quantitative reverse transcription (qRT-PCR) was carried out and the mean male expression of the pk2 gene in the Pel colony in comparison to female expression was 1.6%  of the Pel colony. pk2 expression in pooled testes from 20 Pel males was just detectable but the RT-PCR product was very weak compared to those for wsp and pk1 (not shown).The identical prophage associated ANK encoding genes amed pk2 , showed pk2 , RT-PCR followed by agarose gel electrophoresis revealed similar expression levels in both female and male RNA extracts of the Pel colony. Primers designed to span the intergenic regions of pk2/pk2-1/pk2-2 produced RT-PCR products from females but no detectable products from males of the Pel colony. Primers spanning the intergenic region between pk2 and pk2+1 (primers 5 and 6) produced no amplification of a transcript from either female or male RNA extracts of the Pel colony. However, using the same primers, a product of correct size (733 bp) was amplified in both male and female DNA extracts of the Pel colony.Differential expression between sexes was also observed for two genes directly downstream of 2 Figure . pk2-1 epk2/WD0636 homolog wPip_ANK16 was present in all the Culex strains tested based on PCR amplification. In the Pel, Mol and Sumo Cyppe colonies, wPip_ANK16 was expressed equally in males and females; however for the Col colony no expression could be detected in males, and only a weak RT-PCR product could be detected in females  produced from three prophage associated genes including pk2. However, there was no evidence that wPip_ANK1 and wPip_ANK52 are part of sex-specifically regulated operons.The presence of 60 ANK genes is significantly more than the 23 identified in the l genome ; in factWolbachia, comparing the expression of ankyrin genes to the surface protein encoding gene (wsp) was used to normalize for variation in Wolbachia density but any differences in levels of wsp expression between sexes and stages could be a confounding factor. The wsp gene was previously shown to be expressed in all Cx. pipiens life stages including male and female adults [The quantitative RT-PCR analysis in this study represents only an estimation of differences in relative ANK gene expression. The accurate quantification of RNA expression in bacteria has been limited due to the absence of reliable standardization. In eukaryotic cells, stably expressed housekeeping genes can be used as standards to perform relative quantification of gene expression. For an endosymbiotic bacterium such as e adults .Wolbachia's interaction with the host. A proteomics analysis including experiments such as immunolocalisation studies could be used to characterize the function of ANK proteins in Wolbachia. Current limitations to such studies include the difficulty of obtaining epitope specificity and the absence of a transformation system for Wolbachia. As co-regulated genes are highly likely to show functional interactions, studies to examine the role of the co-expressed prophage-associated genes adjacent to pk2 are also needed.As some of the ANK proteins have predicted signal peptides and transmembrane domains, it is possible that they are secreted into the mosquito cytoplasm or presented on the surface of the bacterium, which could suggest that they are involved in Wolbachia-induced CI in the Cx. pipiens group. The significance of sex-specific expression patterns in the pk2 genes in some host strains but not others is not yet understood, but its occurrence did not correlate with the crossing patterns described in Table pk2 genes in adult mosquitoes. Given the complexity of the phenotype in the Cx. pipiens group, it seems plausible or even probable that the genetic basis for these crossing type differences involves multiple Wolbachia genes, and factors such as the mosquito nuclear background interacting with Wolbachia variants can also contribute [wPip does provide further support for adaptations to sex-specific interactions with its host.Associations between ANK gene sequence variants and particular crossing types have previously been reported , enablinntribute . A hypotwPip genome is the highest yet reported in a prokaryote. The sex-specificity observed in patterns of expression for some of these genes and the differential expression between mosquito strains are also very unusual features, particularly given the generally very high level of sequence conservation between wPip variants. The elucidation of the functional roles and mechanisms of evolution of this family of genes will provide many insights into the biology of reproductive parasites.The number of ANK genes in the wPip genome using ORPHEUS [Wolbachia strain wMel using blastp in order to identify possible homologs. Identification of a putative origin of replication and the assignment of ANK gene numbers was based on the location of the dnaA gene. Gene specific primers with an annealing temperature ranging between 50–55°C were designed for all unique ANK genes , Taq polymerase buffer (1×) and filter-sterilised water in a total volume of 20 μL. Standard PCR cycling conditions involved denaturing the samples for 5 min at 94°C, variable annealing temperature and 72°C (1 min each), followed by a 10 min extension at 72°C using an Applied Biosystems GeneAmp PCR system 2700 . PCR assays were optimised by testing at numerous annealing temperatures.All ANK gene primers were tested on Pel genomic DNA extracted using a modified version of the Livak buffer method with ethanol precipitation . GenomicWolbachia is significantly lower in adult male Culex mosquitoes compared to females, three adult Cx. pipiens male mosquitoes were pooled prior to RNA extraction to increase the amount of Wolbachia RNA present for analysis. RNA extraction of testes was carried out by dissection of 20 Pel colony males under a dissecting microscope in 0.1% saline after immobilising adult mosquitoes on ice. Dissected testes were rinsed in PBS and then pooled in 1.5 mL microcentrifuge tubes in RNAlater  to prevent RNA degradation. The quality and yield of total RNA was measured using a Nanodrop ND 100 spectrophotometer.Total RNA was extracted from young (1–2 days post eclosion) adult mosquitoes using Tri Reagent (Sigma-Aldrich) followed by chloroform extraction and isopropanol precipitation. RNA extracts were treated with DNase I (Sigma-Aldrich) to remove any contaminating DNA. As the density of Taq polymerase only, Sigma-Aldrich) to the reaction mixture were included.Reverse transcription (RT) PCR analysis was performed using the Qiagen Onestep RT-PCR kit . RNase-free water, Onestep RT-PCR buffer (1×), 400 μM dNTPs and Onestep RT-PCR enzyme mix were combined with gene specific primers (0.6 μM) to amplify 2.0 μL of template RNA in 50 μL reactions. Reverse transcription was carried out at 50°C for 30 min followed by 95°C for 15 min. Samples were PCR amplified by denaturing for 5 min at 94°C, cycled 35 times at 94°C (1 min) variable annealing temperature (1 min) and 72°C (1 min each), followed by a 10 min extension at 72°C using an Applied Biosystems GeneAmp PCR system 2700. A total of 10 μL of RT-PCR products and a 100 base-pair marker (Sigma-Aldrich) was electrophoresed on 1% agarose gels stained with ethidium bromide and visualized under ultraviolet illumination. To examine for false positives that might result from amplification of DNA, parallel reactions without adding the reverse transcriptase . Primers were designed to amplify ANK gene fragments of less than 250 bp. Standard curves were produced using serial dilution of RNA extracted from adult female mosquitoes and relative male RNA extract expression of ANK genes measured in comparison. Quantitative RT-PCR cycling conditions were 50°C for 30 min followed by 95°C for 15 min. Samples were cycled 40 times at 94°C (15 s), 55°C (30 s) and 72°C (30 s) followed by a read step. A melting curve was constructed between 50°C and 90°C. Quantitative RT-PCR assays were carried out on six male RNA extracts in two separate assays. Comparing the concentration of cDNA amplified from ankyrin genes to the The author(s) declares that there are no competing interests.TW designed and conducted expression experiments and analyses, LK contributed to primer design, genome analyses and annotation, MS, MJS, NT and JP carried out genome analyses and assembly, and SPS contributed experimental design, co-ordination and analyses. TW, LK and SPS wrote the paper and all authors read and approved the manuscript.wPip ANK gene primers. ANK gene primers, optimal PCR annealing temperatures and PCR product sizes used for reverse transcription PCR analysis. Identical prophage-associated genes at different locations in the genome are listed together.Click here for file


An animal model commonly used to investigate pathways and potential therapeutic interventions relevant to abdominal aortic aneurysm (AAA) involves subcutaneous infusion of angiotensin II within the apolipoprotein E deficient mouse. The aim of this study was to investigate genes differentially expressed in aneurysms forming within this mouse model in order to assess the relevance of this model to human AAA.Using microarrays we identified genes relevant to aneurysm formation within apolipoprotein E deficient mice. Firstly we investigated genes differentially expressed in the aneurysm prone segment of the suprarenal aorta in these mice. Secondly we investigated genes that were differentially expressed in the aortas of mice developing aneurysms relative to those that did not develop aneurysms in response to angiotensin II infusion. Our findings suggest that a host of inflammation and extracellular matrix remodelling pathways are upregulated within the aorta in mice developing aneurysms. Kyoto Encyclopedia of Genes and Genome categories enriched in the aortas of mice with aneurysms included cytokine-cytokine receptor interaction, leukocyte transendothelial migration, natural killer cell mediated cytotoxicity and hematopoietic cell lineage. Genes associated with extracellular matrix remodelling, such as a range of matrix metalloproteinases were also differentially expressed in relation to aneurysm formation.This study is the first report describing whole genome expression arrays in the apolipoprotein E deficient mice in relation to aneurysm formation. The findings suggest that the pathways believed to be critical in human AAA are also relevant to aneurysm formation in this mouse model. The findings therefore support the value of this model to investigate interventions and mechanisms of human AAA. The aim of the current study was to identify genes and pathways associated with aneurysm formation in the angiotensin II infused ApoE-/- mouse model. We used three approaches. Firstly we aimed to identify genes which might underlie the protection of the infrarenal aorta from aneurysm formation in ApoE-/- mice. Secondly we aimed to identify genes which were differentially expressed within the aortas of mice developing aneurysms in comparison to those that did not. Finally we selectively examined whether the differences in gene expression associated with aneurysm formation translated into similar changes in protein expression. The design of these studies is illustrated in Table Abdominal aortic aneurysm (AAA) affects approximately 5% of men and 1% of women aged >60 years . AAA acc animals . This mo animals . In contin vitro and play a vascular protective role in animal models [-/- mice [A total of 26,522 transcripts (73% of the reference list) were expressed above background and compared between segments. A total of 304 transcripts were differentially expressed  between supra and infrarenal aortic segments . A full list of transcripts is given in Additional File l models ,12. Pik3l models . Since v-/- mice . Perhaps-/- mice .(red) or downregulated (green) in expression within the three groups. No change in expression is indicated in black. Hierachical clustering analysis indicated four main patterns of transcript expression , genes encoding markers of inflammatory cells  and many cytokine and chemokine genes  (Table Apoc1 (Apolipoprotein C-I) and Stxbp2 (syntaxin binding protein 2), have been previously demonstrated to be upregulated within biopsies of human AAA [Maximum aortic diameters measured in the aortic arch, thoracic, suprarenal and infrarenal aortas in saline controls and mice exposed to angiotensin II that did and did not develop aneurysms are shown in Table cells Cd1, Cd68 ancells Cd1, Cd68 anIl6, Ccl4, Ccl8 and MMP2 within aortic aneurysms using real time PCR (Table Tnfrsf11b (osteoprotegerin) and Tgfb1 (TGF-b1) genes across the three groups translated to differences in protein expression using samples from study 3. We measured the concentration of OPG and TGFb-1 in the suprarenal segments of angiotensin II treated mice that developed AAA  and those that did not  and found that both proteins were expressed at higher concentrations in aortas of mice with aneurysms, p < 0.05  within the suprarenal aorta of ApoE-/- mice may be relevant to its predilection for aneurysm formation following angiotensin II infusion [In vitro laminin also plays important roles in modulating inflammation and MMP production suggesting its relevance to aneurysm formation [We also investigated genes that might underlie the predilection for suprarenal aneurysm formation within this model, highlighting downregulation of a number of genes which may explain the susceptibility of the suprarenal aorta to aneurysm formation. Urocortin has been shown infusion ,12. We ainfusion ,28. In vormation ,30. FurtA small number of previous studies have used expression arrays to examine gene transcripts associated with AAA in rodent models or patient samples ,28,31-34-/- model. We used an explorative approach using relatively small numbers of arrays . We were not adequately powered to adjust for multiple testing given the number of transcripts examined. Unlike some other microarray studies we have used real time PCR, IHC and ELISA to confirm some of our findings rather than relying on RNA assessment alone [This study is the first to carry out a whole genome expression analysis within the angiotensin II- Apo Ent alone . Numerount alone ,35. Our -/- mouse model for investigating mechanisms and interventions relevant to human AAA.In conclusion our study supports the value of the angiotensin II infused ApoE-/- mice [-/- mice unexposed to angiotensin II (n = 10). 2) We compared RNA expression from whole aortas of 17 week old male ApoE-/- mice which had been exposed to angiotensin II (1.44 μg/kg/min) for 4 weeks where there was clear evidence of aortic aneurysm formation (n = 5) with that of mice failing to develop aneurysms (n = 7) and those exposed to saline infusion (n = 6). 3) We selected 2 genes and 5 cellular pathways identified as upregulated within the aortas of mice with aneurysms for validation using protein assessments including ELISAs and immunohistochemistry (IHC). The selection of genes was based on their association with pathways we had identified from pathway analysis of the data and recognised ability to be able to assess them using ELISAs or IHC. For study 3, twenty-eight additional 17 week old male ApoE-/- mice were infused with angiotensin II for 4 weeks. Ten mice in study 3 died prematurely due to aortic rupture and were excluded from further analysis in order to avoid post-mortem or post rupture effects confounding assessments, leaving 18 animals in this group.Infusion of angiotensin II induces aortic dilatation particularly affecting the suprarenal aorta in ApoE-/- mice ,6,18-21.-/- mice were obtained from Animal Resources Centre, Canning Vale, Western Australia aged 6–8 weeks. Mice were fed standard chow, and at 13 weeks either their aortas were harvested  or angiotensin II  or saline (n = 6 study 2) infusion commenced. Aortic harvesting and osmotic minipump placement  was carried out under ketamine (150 mg/kg i.p.) and xylazine (10 mg/kg i.p.) anaesthesia as previously described [® , and harvested from arch to iliac bifurcation and stored at -80°C for later analysis. For study 3 mice were euthanized, aortas were perfused with PBS and the suprarenal aortic segments divided into 2 and stored separately at -20°C for later IHC and cytokine assessment.This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Ethical approval was obtained from the local institutional committee prior to commencement of the study. Male ApoEescribed . The 28 escribed -21. In sAortas were placed on a black background and digitally photographed . Maximum diameters of the aortic arch, thoracic, suprarenal and infrarenal aorta were determined from the images using computer-aided analysis . Preliminary studies (n = 27) established that these measurements could be repeated with good intra-observer reproducibility .in vitro transcription amplification that allowed incorporation of biotin-labeled nucleotides using the Illumina TotalPrep RNA amplification kit . mRNA samples were assessed for integrity and purity prior to hybridization using the Bioanalyser 2100 with mRNA Nano chips (Agilent Technologies). cRNA of each sample was hybridized to an Illumina Mouse WG-6 V1.1 BeadChip followed by washing, blocking, and streptavidin-Cy3 staining steps, and scanning with a high-resolution Illumina BeadArray reader scanner. The data extraction was performed by using Illumina Bead Studio V2.3.41 software with the output being raw, non-normalized bead summary values. The BeadStudio matrix contains the summarised expression values , standard error of the bead replicates (BEADSTDEV), number of beads used (Avg_NBEADS) and a detection score, which estimates the confidence limit of detection of a gene. The performance of the built-in controls that accompany each Illumina beadchip experiment was assessed as part of the Bead Studio V2 experiment performance report and was found to be satisfactory. Controls included Housekeeping controls for intactness of the biological specimens, negative controls to establish gene expression detection limits and hybridization controls including low and high stringency controls and biotin signal generation controls. The raw data matrix extracted from Beadstudio was uploaded into GeneSpring GX 7.3.1  software for downstream analysis. Details of GeneSpring analysis including data transformations, per chip and per gene normalisations are described below in Analysis and Design.Total RNA was extracted from homogenised aortas using the TRI reagent (Sigma) and RNeasy Mini kits (Qiagen) and analysed on the HP 2100 Bioanalyzer (Agilent Technologies) for integrity using Eukaryotic total RNA nano chips (Agilent)  using real time PCR. RNA samples from mice exposed to angiotensin II that developed aneurysms (n = 5), mice exposed to angiotensin II that did not develop aneurysms (n = 7) and saline control mice (n = 6) were included in the analysis. The QuantiTect SYBR Green one-step RT-PCR Kit (Qiagen) and Quantitect Primer Assays (Qiagen)  were used according to the manufacturer's instructions with 25 ng of total RNA as template. Primers for mouse Gapdh were used to amplify the housekeeping gene . Standard curves for each gene including the housekeeping gene were constructed using duplicate sets of five (5) 10-fold serial dilutions of equal volumes of the pooled saline control RNAs. Negative and "minus-RT" controls were also included. All reactions were independently repeated in duplicate to ensure the reproducibility of the results. Cycling parameters were as follows: 50°C 30 min for RT; 95°C 15 min; 40 cycles of 94°C 15 sec, 55°C 30 sec, 72°C 30 sec. Data were viewed and analysed using the Rotor-Gene's real-time analysis software . The relative expression of the gene of interest in each sample was calculated by the Rotor-Gene software using the concentration-Ct-standard curve method and normalised using the average expression of Gapdh for each sample. SSPS statistical software was used to calculate median and interquartile ranges for expression of each gene of interest in each group.Using RNA obtained for the mice employed in the micro-array investigations in study 2 we validated findings for 4 genes , and goat anti-rat HRP (Millipore AP136P). Rat IgG (Sigma I4131) was used as isotype control. Detection of osteoprotegerin (OPG) was carried out using biotinylated goat anti-mouse OPG (R&D BAF459) diluted to 1 μg/ml in blocking buffer followed by Vectastain Elite ABC-HRP (Vector Laboratories). Biotinylated goat-IgG was used as isotype control (Vector Laboratories VEBI1001). T cells , B cells , dendritic cells  and neutrophils (Ly6G/Ly6C(Gr-1) FITC clone RB6-8C5, BD Biosciences 553127) were demonstrated using either anti-FITC HRP (Invitrogen), biotinyl-tyramide (Perkin Elmer), SA-HRP (Perkin Elmer) for FITC-conjugated antibodies or SA-HRP, biotinyl-tyramide, SA-HRP for biotinylated antibodies. Appropriate isotype controls were added to other sections . Slides were incubated in the peroxidase substrate 3, 3'-diamminobenzidine , counterstained in Mayer's Haematoxylin, dehydrated, cleared in xylene and mounted in Depex mounting medium. Sections were photographed using a Nikon Eclipse 50i microscope, Digital Sight camera and NIS-elements software.Serial cryostat sections 7 μm thick were cut from suprarenal aortas with and without aneurysms prior to staining for inflammatory cells. Serial frozen sections were air-dried, fixed in acetone for 10 min at -20°C, air dried and rehydrated with PBS before being incubated in 3% Hg at 4°C for 20 min. Supernatant protein was quantified by the Bradford technique . Concentrations of OPG and transforming growth factor beta-1 (TGFb-1) were assessed using commercial ELISAs  and expressed as pg/mg of protein. Initial tests were performed to determine an appropriate amount of extracted aortic protein to load in each well to suit the ELISA standard curves . Serum and extracted aortic proteins from OPG deficient mice were confirmed as having zero readings using the OPG ELISA (data not shown). Batch analysis of the samples was carried out to facilitate comparison between groups. We have previously reported excellent reproducibility of similar assays [Protein was extracted from individual frozen suprarenal aortic segments by homogenising in buffer  and centrifuging at 18,000 × r assays .) and are accessible through GEO accession number GSE7006 (GEO Accession viewer) for study 1 and GSE12591  for study 2. For study 1 which used paired samples, normalisations were as follows: each chip was normalised to the median intensity of the array; each gene was normalised relative to the suprarenal sample of each pair. Thus the expression of each SRA sample was normalised to 1. Volcano plots were used to identify differentially expressed genes with >1.5 fold difference in expression and p < 0.05. Statistical analysis was carried out using a paired t-test  comparing the mean infrarenal/suprarenal ratio to a baseline value of 1 for each sample pair. In study 2 we followed the standard normalization procedures recommended for the GeneSpring GX 7.3.1 software for one-colour array data. In brief, data transformation was corrected for a low signal, with values recorded at <0.01 increased to the minimum (0.01). Default settings were used for experiment normalisation which included chip normalisation to 50th percentile and gene normalisation to the median. The cross gene error model was used to further filter low quality data. We sought to identify genes with a 2-fold differential expression within the aortas of mice with and without aneurysms based on an uncorrected p value of < 0.05 included in our sample size estimate. We initially compared findings from mice that received angiotensin II and developed AAAs (n = 5) with both other groups of mice (n = 7 and 6). Genes showing a greater than 2 fold difference in expression between groups  p < 0.05), were considered to be differentially expressed. We reasoned that the most significant findings would relate to genes differentially expressed between angiotensin II perfused mice, that did (n = 5) and did not (n = 7) develop aneurysms. Volcano plots were used to identify differentially expressed genes between the aortas of these two groups of mice. This group of 531 genes was further investigated by hierarchical clustering in GeneSpring, in which a tree of transcripts or genes is built by successively finding the two most similar gene expression patterns from the full transcript set. Individual samples were then arranged in a condition tree according to overall similarity. We also included the saline group in this clustering analysis in order to show how these 531 genes are expressed in control aortas. Genes with similar expression patterns were grouped as hierarchical clusters with distances between samples computed using Pearson correlations for similarity measures and average linkage as the clustering algorithm. Hierachical clustering revealed several major nodes in the gene tree structure which indicated at least 4 different patterns of gene expression. Lists of differentially expressed genes were examined for biologically relevant associations using Gene Ontologies and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis in the web-based software Webgestalt . Quantitative real time PCR outcomes in study 2 and concentrations of cytokines measured in study 3 were compared between groups using Mann Whitney U test.We designed the current study as an exploratory analysis expecting a minimal level of false discovery inevitable in analysis of large microarray gene probes. Sample size calculations were conducted using SPCalc ,38. For CR carried out the expression arrays, analysis of results and immunohistochemistry, and participated in drafting the manuscript. MN carried out the mice work. AT carried out the real time PCR. BC carried out the ELISAs, analysed these results and participated in drafting the manuscript. JVM participated in drafting and editing the manuscript. JG conceived the study, obtained funding, supervised the project and wrote the main manuscript. All authors read and approved the final manuscript.List of transcripts differentially expressed in supra and infrarenal aortas. Transcripts included were expressed at 1.5 fold different levels and significant at uncorrected p < 0.05. Included are genebank and unigene names, uncorrected p values and ratio of transcripts expression compared between supra and infrarenal aortas.Click here for fileNumber of differentially expressed transcripts comparing aortas of mice with and without aneurysms, in addition to saline controls. Transcripts included were expressed at 2 fold different levels and significant at uncorrected p < 0.05.Click here for fileList of transcripts differentially expressed in aortas of mice exposed to angiotensin II which did (AAA group) and did not (no AAA group) develop aneurysms (n = 531). We considered this the most important comparison since the controls were in all ways similar to the group of interest except they did not have AAAs. Transcripts included were expressed at 2 fold different levels and significant at uncorrected p < 0.05. Transcripts with higher expression in AAA group are indicated in yellow, whilst those with higher expression in no AAA group are coloured blue. Included are gene symbol (where an identified gene is associated with the transcript), genebank accession number, fold difference, uncorrected p values, gene description, and Illumina Probe identification number.Click here for fileList of transcripts differentially expressed in aortas of mice exposed to angiotensin II which developed aneurysms (AAA) and saline controls (n = 1196). Transcripts included were expressed at 2 fold different levels and significant at uncorrected p < 0.05. Included are gene symbol (where an identified gene is associated with the transcript), genebank accession number, fold difference, uncorrected p values, gene description, and Illumina Probe identification number.Click here for fileList of transcripts differentially expressed in aortas of mice exposed to angiotensin II which did not develop aneurysms (no AAA) and saline controls (n = 654). Transcripts included were expressed at 2 fold different levels and significant at uncorrected p < 0.05. Included are gene symbol (where an identified gene is associated with the transcript), genebank accession number, fold difference, uncorrected p values, gene description, and Illumina Probe identification number.Click here for fileList of pattern 1 transcripts (n = 104) which were downregulated with the aortas of aneurysm resistant mice . Included are gene symbol (where an identified gene is associated with the transcript), genebank accession number, uncorrected p values, gene description, Illumina Probe identification number and fold difference comparing aortas of mice exposed to angiotensin II that did (AAA) and did not develop aneurysms (no AAA).Click here for fileList of pattern 2 transcripts which were upregulated in aortic aneurysms . Included are gene symbol (where an identified gene is associated with the transcript), genebank accession number, uncorrected p values, gene description, Illumina Probe identification number and fold difference compared to aortas of mice with no aneurysm (no AAA).Click here for fileList of genes which were downregulated in the aortas of mice exposed to angiotensin II which did not develop aneurysms (no AAA) by comparison to both mice developing aneurysms (AAA) and also saline controls (n = 10). The list of genes was generated from pattern 3 transcripts excluding those that were associated with no known gene or which were not significantly different  by comparison to saline controls in addition (n = 7). These genes potentially have a pathological role in AAA. Genes highlighted with † have previously been identified as upregulated within human AAA biopsies in a whole genome expression study [on study . IncludeClick here for fileList of transcripts which were upregulated in the aortas of mice resistant to aneurysm formation (n = 85). Transcripts highlighted in yellow are increased  in the no AAA group when compared to both AAA and saline groups. These genes are potentially protective role against AAA. Included are gene symbol (where an identified gene is associated with the transcript), genebank accession number, uncorrected p values, gene description, Illumina Probe identification number and fold differences when comparing aortas resistant to aneurysm formation to aortas with aneurysms and saline control aortas.Click here for fileWebgestalt analysis of transcripts upregulated in aortas of mice with aneurysms compared to those without. Both groups of mice received angiotensin II. Of 376 recognised transcripts 302 were known genes and 116 were assigned to one or more KEGG pathways. Included are gene symbol (where an identified gene is associated with the transcript), gene description, Illumina Probe identification number and KEGG title.Click here for fileExamples of mouse RNA samples examined on the Agilent Bioanalyser. The 10 pooled samples  used in study 1 (with repetition of 1 sample) were analysed in comparison to molecular weight markers (far left), 18 and 28S RNA controls (far right). All samples had RNA integrity scores of between 7.5 and 8.1.Click here for file-/- mice exposed to angiotensin II or saline subcutaneously for 4 weeks from study 2Digital photographs of aortas from 17 week old male ApoE. A) Example of an aorta from a mouse in which an aneurysm developed in response to angiotensin II infusion. B) Example of an aorta from a mouse in which an aneurysm did not develop in response to angiotensin II infusion. C) Example of an aorta from a mouse exposed to saline infusion for 4 weeks with no aneurysm formation.Click here for file