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Quantitative RT-PCR (quantitative + rt-pcr)
Kinds of Quantitative RT-PCR Terms modified by Quantitative RT-PCR Selected AbstractsIdentification of transcriptional targets associated with the expression of p210 Bcr-AblEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2006Fionnuala B. Hickey Abstract:,Objectives:,Chronic myeloid leukaemia is caused by the expression of the p210 Bcr-Abl fusion protein which results from the Philadelphia translocation, t(9;22). This oncogene has been the focus of extensive research. However, the molecular mechanisms responsible for the haematological malignancy are not fully understood. The main objective of the current study was to identify novel transcriptional targets of Bcr-Abl. Methods:,In order to achieve this, microarrays were employed in order to conduct a genome-wide expression analysis comparing 32D cells with a transfected clone expressing high levels of p210 Bcr-Abl. Quantitative RT-PCR was employed in order to confirm the observed increase/decrease in expression for a number of the deregulated genes. Results and conclusions:,This comparison identified 138 genes of known function showing altered expression in response to Bcr-Abl-mediated signalling. Among the genes found to be upregulated in response to p210 Bcr-Abl were aldolase 1A and phosphofructokinase, both of which encode key enzymes in the glycolytic pathway. As a consequence of this, we demonstrate that the rate of glycolysis is significantly increased in Bcr-Abl expressing cells in a PI3K-dependent manner. Our results also indicate altered expression of genes involved in cell proliferation, cell adhesion and cell signalling. [source] Gene Expression Profiling in Cluster Headache: A Pilot Microarray StudyHEADACHE, Issue 10 2006Christina Sjöstrand MD Background.,Cluster headache (CH) is a primary neurovascular headache disorder characterized by attacks of excruciating pain accompanied by ipsilateral autonomic symptoms. CH pathophysiology is presumed to involve an activation of hypothalamic and trigeminovascular systems, but inflammation and immunological mechanisms have also been hypothesized to be of importance. Objective.,To identify differentially expressed genes during different clinical phases of CH, assuming that changes of pathophysiological importance would also be seen in peripheral venous blood. Methods.,Blood samples were drawn at 3 consecutive occasions from 3 episodic CH patients: during attacks, between attacks and in remission, and at 1 occasion from 3 matched controls. Global gene expression was analyzed with microarray tehnology using the Affymetrix Human Genome U133 2.0 Plus GeneChip® Set, covering more than 54,000 gene transcripts, corresponding to almost 22,000 genes. Quantitative RT-PCR on S100P gene expression was analyzed in 6 patients and 14 controls. Results.,Overall, quite small differences were seen intraindividually and large differences interindividually. However, pairwise comparisons of signal values showed upregulation of several S100 calcium binding proteins; S100A8 (calgranulin A), S100A12 (calgranulin C), and S100P during active phase of the disease compared to remission. Also, annexin A3 (calcium-binding) and ICAM3 showed upregulation. BIRC1 (neuronal apoptosis inhibitory protein), CREB5, HLA-DQA1, and HLA-DQB1 were upregulated in patients compared to controls. The upregulation of S100P during attack versus remission was confirmed by quantitative RT-PCR analysis. Conclusions.,The S100A8 and S100A12 proteins are considered markers of non-infectious inflammatory disease, while the function of S100P is still largely unknown. Furthermore, upregulation of HLA-DQ genes in CH patients may also indicate an inflammatory response. Upregulation of these pro-inflammatory genes during the active phase of CH has not formerly been reported. Data from this pilot microarray study provide a basis for further studies in CH. [source] Transcriptional profiling on chromosome 19p indicated frequent downregulation of ACP5 expression in hepatocellular carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 6 2005Kathy Y.-Y. Abstract Chromosomal rearrangements unraveled by spectral karyotyping (SKY) indicated frequent chromosome 19 translocations in hepatocellular carcinoma (HCC). In an effort to characterize the aberrant 19 rearrangements in HCC, we performed positional mapping by fluorescence in-situ hybridization (FISH) in 10 HCC cell lines. SKY analysis indicated structural rearrangements of chromosome 19 in 6 cell lines, 4 of which demonstrated recurring 19p translocations with different partner chromosomes. Using fluorescence-labeled BAC probes, physical mapping indicated a breakpoint cluster between 19p13.12 and 19p12. A corresponding transcriptional mapping by cDNA array on 19p suggested the differential expression of a single downregulated gene ACP5 (tartrate-resistant acid phosphatase type 5). Quantitative RT-PCR confirmed the reduced expression of ACP5 and indicated a strong correlation of its repressed expression only in cell lines that contain a 19p rearrangement (p = 0.004). We further examined the expression of ACP5 in a cohort of 82 primary tumors and 74 matching nonmalignant liver tissues. In the primary HCC examined, a reduction of ACP5 transcripts by 2 to as much as 1,000-fold was suggested in 67% of tumors (55/82 cases). When compared to adjacent nonmalignant tissues, 46% of tumors (34/74 cases) demonstrated a lower expression level (p = 0.015). On closer examination, a high significance of ACP5 repression was suggested in the cirrhotic HCC subgroup that was derived from chronic hepatitis B infected patients (55%; 30/54 cases; p = 0.001). Functional examination of ACP5 ectopic expression in HCC cells further demonstrated a significant growth inhibitory effect of ACP5 on tumor cell survival (p < 0.001). In our study, the novel finding of common ACP5 downregulation in HCC may provide basis for further investigations on the role of acid phosphatase in hepatocarcinogenesis. © 2004 Wiley-Liss, Inc. [source] Overexpression of hns in the plant growth-promoting bacterium Enterobacter cloacae UW5 increases root colonizationJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2010M.M. English Abstract Aims:, Plant growth-promoting rhizobacteria (PGPR) introduced into soil often do not compete effectively with indigenous micro-organisms for plant colonization. The aim of this study was to identify novel genes that are important for root colonization by the PGPR Enterobacter cloacae UW5. Methods and Results:, A library of transposon mutants of Ent. cloacae UW5 was screened for mutants with altered ability to colonize canola roots using a thermal asymmetric interlaced (TAIL)-PCR-based approach. A PCR fragment from one mutant was reproducibly amplified at greater levels from genomic DNA extracted from mutant pools recovered from seedling roots 6 days after seed inoculation compared to that from the cognate inoculum cultures. Competition assays confirmed that the purified mutant designated Ent. cloacae J28 outcompetes the wild-type strain on roots but not in liquid cultures. In Ent. cloacae J28, the transposon is inserted upstream of the hns gene. Quantitative RT-PCR showed that transposon insertion increased expression of hns on roots. Conclusions:, These results indicate that increased expression of hns in Ent. cloacae enhances competitive colonization of roots. Significance and Impact of the Study:, A better understanding of the genes involved in plant colonization will contribute to the development of PGPR that can compete more effectively in agricultural soils. [source] The sodium pump ,1 sub-unit: a disease progression,related target for metastatic melanoma treatmentJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Véronique Mathieu Abstract Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump , sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump ,1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump , sub-units in melanoma clinical samples and cell lines and also to characterize the role of ,1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump , sub-units. In vitro cytotoxicity of various cardenolides and of an anti-,1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the ,1 sub-unit, and 33% of human melanomas displayed significant ,1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The ,1 sodium pump sub-unit could represent a potential novel target for combating melanoma. [source] Gene regulation of ,4,2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effectsJOURNAL OF NEUROCHEMISTRY, Issue 3 2009Vishnu Hosur Abstract ,4,2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of ,4,2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 ,M nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in h,4,2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in 3[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1, and IL-6. Nicotine suppresses IL-1, and IL-6 expression at least in part by inhibiting NF,B activation. Antagonists dihydro-,-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing ,4,2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal ,4,2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of ,4,2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate ,4,2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic ,4,2 receptor activation promotes anti-inflammatory effects similar to ,7 receptor activation. [source] Bone morphogenetic proteins 4, 6, and 7 are up-regulated in mouse spinal cord during experimental autoimmune encephalomyelitisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2008Jahan Ara Abstract Although spontaneous remyelination occurs in multiple sclerosis (MS), the extent of myelin repair is often inadequate to restore normal function. Oligodendrocyte precursors remaining in nonremyelinating MS plaques may be restricted by an inhibitory signal. Bone morphogenetic proteins (BMPs) have been implicated as repressors of oligodendrocyte development and inducers of astrogliogenesis. We hypothesized that BMPs are up-regulated in MS lesions and play a role in demyelination and astrogliosis. We examined expression of BMPs in an animal model of MS, chronic experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide in C57BL/6 mice. By 14 days postimmunization, compared to those of control mice, the lumbar spinal cords of MOG-peptide EAE mice demonstrated prominent astrogliosis, infiltration of inflammatory cells, and disrupted expression of myelin proteins. Quantitative RT-PCR showed that expression of BMP4, BMP6, and BMP7 mRNA increased 2- to 4-fold in the lumbar spinal cords of animals with symptomatic EAE versus in vehicle-treated and untreated controls on days 14, 21, and 42 postimmunization. BMP2 mRNA expression was not altered. BMP4 mRNA was much more abundant in the spinal cords of all animals than was mRNA encoding BMP2, BMP6, and BMP7. Immunoblot analysis confirmed the increased expression of BMP4 in the EAE animals. Immunohistochemistry revealed increased BMP4 immunoreactivity in areas of inflammation in MOG-peptide EAE animals. BMP4 labeling was mostly limited to macrophages but was sometimes associated with astrocytes and oligodendrocytes. These results indicate that members of the BMP family are differentially expressed in adult spinal cord and are up-regulated during EAE. © 2007 Wiley-Liss, Inc. [source] Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewageLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2004M.A. Laverick Abstract Aims:, Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. Methods and Results:, Noroviruses in environmental water samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler® and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3,-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. Conclusions:, Sewage was found to contain up to 1·8 × 106 norovirus cDNA copies per 100 ml and effluent contained up to 1·7 × 106 copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. Significance and Impact of the Study:, The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods. [source] Expression and function of phosphodiesterases in nitrofen-induced congenital diaphragmatic hernia in ratsPEDIATRIC PULMONOLOGY, Issue 4 2010Irene W.J.M. van der Horst MD Abstract Background Congenital diaphragmatic hernia (CDH) is an anomaly associated with pulmonary hypoplasia and pulmonary hypertension (PH). The limited efficacy of current approaches to treat PH in CDH, including inhaled nitric oxide (NO), drives the search for other therapies. Phosphodiesterases (PDEs) degrade cyclic nucleotide second messenger cAMP and cGMP downstream of NO thereby limiting the vasodilatory response to NO. Objective To identify therapeutic targets by cataloguing the expression and function of PDE isoforms in the pulmonary vasculature in nitrofen-induced CDH in fetal rats. Methods/Results Quantitative RT-PCR revealed PDE1,5 and PDE9 mRNA expression in pulmonary arteries (PAs) of control and nitrofen-induced CDH term fetal rats. In this order of potency, the PDE inhibitors Sildenafil (PDE5),>,EHNA (PDE2),>,Rolipram (PDE4),>,Cilostamide (PDE3) all dilated isolated third generation PA after pre-constriction with the thromboxane analog U46619. Hyperoxic pre-incubation of PAs significantly attenuated vasodilatation induced by the PDE5 inhibitor Sildenafil (65% vs. 33%, P,<,0.004). CDH PAs dilated significantly less to PDE2 inhibitor EHNA compared to control (51% vs. 72%, P,<,0.05). Subsequently PDE2 protein expression was higher in PAs of CDH animals. Conclusion Most PDE isoforms exist in the PAs of fetal rats and their inhibition causes pulmonary vasodilatation. PDE5 inhibition was the most potent vasodilator, however, there were no differences between groups. PDE5-induced vasodilatation was attenuated by hyperoxic pre-incubation. PDE inhibitors might be considered therapeutic targets in combination with iNO in neonates with CDH. Pediatr Pulmonol. 2010; 45:320,325. © 2010 Wiley-Liss, Inc. [source] Effect of Rds abundance on cone outer segment morphogenesis, photoreceptor gene expression, and outer limiting membrane integrityTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 6 2007Rafal Farjo Abstract We examined the molecular, structural, and functional consequences on cone photoreceptors of the neural retinal leucine zipper knockout (Nrl,/,) mice when only one allele of retinal degeneration slow (Rds) is present (Rds+/,/Nrl,/,). Quantitative RT-PCR and immunoblot analysis were used to assess the expression levels of several phototransduction genes; electroretinography was used to assess quantitatively the retinal responsiveness to light; and immunohistochemistry and ultrastructural analysis were used to examine retinal protein distribution and morphology, respectively. In Rds/Nrl double-null mice, S-cones form dysmorphic outer segments that lack lamellae and fail to associate properly with the cone matrix sheath and the outer limiting membrane. In Rds+/,/Nrl,/, mice, cones form oversized and disorganized outer segment lamellae; although outer limiting membrane associations are maintained, normal interactions with cone matrix sheaths are not, and photoreceptor rosette formation is observed. These retinas produce significantly higher photopic a-wave and b-wave amplitudes than do those of Rds,/,/Nrl,/, mice, and the levels of several cone phototransduction genes are significantly increased coincidently with the presence of Rds and partial lamellae formation. Thus, as in rod photoreceptors, expression of only one Rds allele is unable to support normal outer segment morphogenesis in cones. However, cone lamellae assembly, albeit disorganized, concomitantly permits outer limiting membrane association, and this appears to be linked to photoreceptor rosette formation in the rodless (cone-only) Nrl,/, retina. In addition, photoreceptor gene expression alterations occur in parallel with changes in Rds levels. J. Comp. Neurol. 504:619,630, 2007. © 2007 Wiley-Liss, Inc. [source] Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissueTHE PROSTATE, Issue 8 2009Thomas J. Walton Abstract BACKGROUND Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ER,) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. METHODS Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ER,), estrogen receptor alpha (ER,), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann,Whitney U -test. Correlation coefficients were analyzed using Spearman's test. RESULTS Significant positive correlations were seen when AR and AR-dependent PSA, and ER, and ER,-dependent PGR were compared, indicating a representative population of RNA transcripts. ER, gene expression was significantly over-expressed in the cancer group compared with benign controls (P,<,0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P,<,0.05). There were no significant differences in AR, ER, or PSA expression between the groups. This study represents the first to show an upregulation of ER, gene expression in laser microdissected prostate cancer specimens. CONCLUSIONS In concert with recent studies the findings suggest differential production of ER, splice variants, which may play important roles in the genesis of prostate cancer. Prostate 69: 810,819, 2009. © 2009 Wiley-Liss, Inc. [source] Expression of CCL5 (RANTES) and CCR5 in prostate cancer,THE PROSTATE, Issue 2 2006Gayle G. Vaday Abstract Background Expression of the inflammatory chemokine CCL5 (RANTES) by tumor cells is thought to correlate with the progression of several cancers. CCL5 was shown to induce breast cancer cell migration, mediated by the receptor CCR5. A CCR5 antagonist was demonstrated to inhibit experimental breast tumor growth. Recently, CCL5 and CCR5 mRNA expression was reported in prostate cancer (PCa) tissues. Herein, we characterized CCL5 and CCR5 expression in cultures of PCa cells and explored possible functions of CCL5 in PCa progression. Methods Quantitative RT-PCR, ELISA, and immunohistochemical staining were performed to examine CCL5 expression in prostate cell lines. CCR5 expression was measured by flow cytometry. Proliferation and invasion assays were performed to determine potential functions of CCL5 and CCR5 in PCa. Results Expression of CCL5 mRNA and protein was found in human PCa cell lines (PC-3; DU-145; LNCaP) and primary prostate adenocarcinoma cells. CCL5 and CCR5 were also detected in human PCa tissues. CCR5 expression was demonstrated on the cell surface of PCa cells, as well as in intracellular pools. Incubation with CCL5 (10,100 ng/ml) induced PCa cell proliferation, and the CCR5 antagonist TAK-779 inhibited CCL5-induced proliferation. CCL5 was found to stimulate PCa cell invasion, and TAK-779 blocked the effects of CCL5. Conclusions In light of evidence that inflammation influences the pathogenesis of PCa, these results suggest that inflammatory chemokines, such as CCL5, expressed by prostate cells may act directly on the growth and survival of PCa cells. Chemokine receptor antagonists may thus block autocrine mechanisms of PCa progression. Published 2005 Wiley-Liss, Inc. [source] Endothelin receptor B2 (EDNRB2) is associated with the panda plumage colour mutation in Japanese quailANIMAL GENETICS, Issue 2 2007M. Miwa Summary The panda mutant in Japanese quail (Coturnix japonica) displays spots of wild-type plumage on a white background and is controlled by an autosomal recessive allele (s). The dotted white is controlled by a third allele (sdw) of the s locus with sdw/sdw quail having less pigmentation than s/s quail. We mapped the s locus to the Japanese quail chromosome 4 (CJA04) in a previous study. The orthologous region of the chicken (Gallus gallus) genome includes endothelin receptor B2 (EDNRB2), an avian-specific paralog of endothelin receptor B (EDNRB). EDNRB mutations in mammals retard the migration of neural crest cells (NCCs), which results in a spotted coat colour and an enteric nervous defect. In the present study, we investigated the association between the s locus and EDNRB2 in Japanese quail. Sequence comparison among transcripts from livers of wild-type, panda and dotted white quail revealed a nucleotide substitution (c.995G>A) leading to a p.R332H amino acid change that was specific to panda, whereas no amino acid substitution was found in dotted white birds. The amino acid position 332 is located in the sixth transmembrane domain and is highly conserved in both avian and mammalian endothelin receptors. The A allele at nucleotide position 995 was specific to panda (s/s) birds among 10 strains, and was mapped to the same chromosomal region as the s locus. Quantitative RT-PCR revealed that EDNRB2 transcripts were reduced in both panda and dotted white mutants compared with wild-type. However, there was no difference between the early embryos of wild-type and panda with respect to the migration of NCCs. The genetic association of EDNRB2 with plumage colour in birds was found for the first time in this study. [source] Target of rapamycin in the oriental fruit fly Bactrocera dorsalis (hendel): its cloning and effect on yolk protein expressionARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010R. Suganya Abstract Target of rapamycin (TOR), a serine/threonine protein kinase, is involved in regulating a number of growth and developmental processes of an organism, including yolk protein synthesis in insects. In this study, TOR gene was isolated, designated BdTOR (GenBank accession no. FJ167395), from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed a higher expression of BdTOR in the pupa than in other developmental stages, as well as in ovary than in the fat body. Downregulation of BdTOR activity by rapamycin treatment and RNA interference (RNAi) in vivo resulted in a significant reduction in yolk protein transcripts in both fat body and ovary, with a substantial reduction in ovary size. However, an unexpected increase in the expression of yolk protein gene was observed in adult ovary 9 days after rapamycin treatment. Taken together, the results suggest the involvement of BdTOR in the regulation of yolk protein synthesis in B. dorsalis. © 2010 Wiley Periodicals, Inc. [source] Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogsBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2009K.M. Credille Summary Background, Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. Objectives, To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. Methods, Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. Results, Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. Conclusions, Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion. [source] Developmental toxicity of estrogenic chemicals on rodents and other speciesCONGENITAL ANOMALIES, Issue 2 2002Taisen Iguchi ABSTRACT, Antenatal sex-hormone exposure induces lesions in mouse reproductive organs, which are similar to those in humans exposed in utero to a synthetic estrogen, diethylstilbestrol. The developing organisms including rodents, fish and amphibians are particularly sensitive to exposure to estrogenic chemicals during a critical window. Exposure to estrogens during the critical period induces long-term changes in reproductive as well as non-reproductive organs, including persistent molecular alterations. The antenatal mouse model can be utilized as an indicator of possible long-term consequences of exposure to exogenous estrogenic compounds including possible environmental endocrine disrupters. Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans, some of which exhibit estrogenic activity by binding to the estrogen receptors. Estrogen responsive genes, therefore, need to be identified to understand the molecular basis of estrogenic actions. In order to understand molecular mechanisms of estrogenic chemicals on developing organisms, we are identifying estrogen responsive genes using cDNA microarray, quantitative RT-PCR, and differential display methods, and genes related to the estrogen-independent vaginal changes in mice induced by estrogens during the critical window. In this review, discussion of our own findings related to endocrine distuptor issue will be provided. [source] Transient expression of serotonin 5-HT4 receptors in the mouse developing thalamocortical projectionsDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2010Erin R. Slaten Abstract The serotonin 5-HT4 receptor (5-HT4 -R) is an unusually complex G-protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5-HT4 -Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT-PCR. The developing thalamocortical projections transiently expressed 5-HT4 -Rs in the embryonic brain and the 5-HT4 -R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13,17, the forebrain mRNA levels of the 5-HT4(a) -R and 5-HT4(b) -R splice variants increased nine- and fivefold, respectively, whereas the levels of the 5-HT4(e) -R and 5-HT4(f) -R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5-HT4 -R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010. [source] Sex differences in and hormonal regulation of Kv1 potassium channel gene expression in the electric organ: Molecular control of a social signalDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2007W. Preston Few Abstract Electric fish communicate with electric organ (EO) discharges (EODs) that are sexually dimorphic, hormone-sensitive, and often individually distinct. The cells of the EO (electrocytes) of the weakly electric fish Sternopygus possess delayed rectifying K+ currents that systematically vary in their activation and deactivation kinetics, and this precise variation in K+ current kinetics helps shape sex and individual differences in the EOD. Because members of the Kv1 subfamily produce delayed rectifier currents, we cloned a number of genes in the Kv1 subfamily from the EO of Sternopygus. Using our sequences and those from genome databases, we found that in teleost fish Kv1.1 and Kv1.2 exist as duplicate pairs (Kv1.1a&b, Kv1.2a&b) whereas Kv1.3 does not. Using real-time quantitative RT-PCR, we found that Kv1.1a and Kv1.2a, but not Kv1.2b, expression in the EO is higher in high EOD frequency females (which have fast EO K+ currents) than in low EOD frequency males (which have slow EO K+ currents). Systemic treatment with dihydrotestosterone decreased Kv1.1a and Kv1.2a, but not Kv1.2b, expression in the EO, whereas treatment with human chorionic gonadotropin (hCG) increased Kv1.2a but not Kv1.1a or Kv1.2b expression in the EO. Thus, systematic variation in the ratios of Kv1 channels expressed in the EO is correlated with individual differences in and sexual dimorphism of a communication signal. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Quantitative assessment of WT1 gene expression after allogeneic stem cell transplantation is a useful tool for monitoring minimal residual disease in acute myeloid leukemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2009Anna Candoni Abstract Introduction:,WT1 overexpression is described in several oncological diseases including acute myeloid leukemia (AML). Quantification of WT1 in bone marrow samples may be useful as a marker of minimal residual disease (MRD) and may predict the relapse of AML after allogeneic hematopoietic stem cell transplant (HSCT). Methods and results:, The quantitative expression of WT1 was measured in 38 AML patients (16 males and 22 females) at diagnosis, at the time of transplant and after the allogeneic HSCT (at precise time points). All cases showed high WT1 expression levels at diagnosis with a mean of 4189 (SD 3325) and a median of 3495 (range 454,13923) copies WT1/104Abl. At transplant, 25 patients (66%) were in complete cytologic remission (CcR) and 13 (34%) had refractory or relapsed AML. Bone marrow samples from patients transplanted in CcR showed significantly lower WT1 expression levels during HSCT compared with the samples from patients with a relapsed or refractory AML (P = 0.004). After HSCT, a rapid decline in WT1 expression levels was observed in all patients who attained or maintained a condition of CcR. Six of 38 patients (13%) relapsed after HSCT and all of them had an increase in WT1 expression at/or before relapse. Five of these six patients died of leukemia and one was successfully reinduced with donor lymphocyte infusion (DLI) + chemotherapy with a rapid reduction of WT1 levels. Besides, we found a complete concordance between WT1 expression levels and other disease markers (when available). Conclusions:, In our experience, there was a complete concordance between WT1 expression levels (measured by quantitative RT-PCR at precise time points) and status of AML before and after allogeneic HSCT. WT1 may be useful as a non-specific leukemia marker for monitoring MRD and as a predictor of AML clinical relapse. Based on these results, cases with increase of WT1 levels after HSCT and without graft vs. host disease may be candidate to discontinuation of immunosuppression and/or DLI therapy. [source] CLLU1 expression levels predict time to initiation of therapy and overall survival in chronic lymphocytic leukemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2006Anne Mette Buhl Abstract:,Objectives:,Chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. IgVH mutational status, chromosomal aberrations, CD38 expression and ZAP-70 expression are prognostic markers in CLL, however, they are not exclusively confined to this disease. We recently identified a novel CLL-specific gene (CLL upregulated gene1, CLLU1) that is exclusively upregulated in CLL cells. Here we describe our evaluation of the prognostic significance of CLLU1 in CLL. Methods:,A cohort of 59 previously untreated CLL patients was studied. We determined the expression levels of two CLLU1 transcripts, cDNA1 and CDS, by quantitative RT-PCR. The relation between CLLU1 expression and time to therapy, overall survival and presence or absence of ZAP-70, CD38, chromosomal aberrations or IgVH mutations in the 59 patients was analyzed. Results:,Analyzed as a continuous, quantitative parameter CLLU1 levels significantly predicted time from diagnosis to initiation of therapy (P , 0.0003) Analyzed as a categorical parameter, by segregation of the patients into groups with cDNA1 or CDS expression above or below the median, the CLLU1 levels significantly predicted time from diagnosis to initiation of therapy (P = 0.001) and predicted overall survival with borderline significance (P , 0.05). Patient stratification according to clinical stage, cytogenetics, IgVH mutational status, ZAP-70 and CD38, demonstrated significantly increased CLLU1 expression in all investigated CLL poor risk groups. CLLU1 expression levels contributed additional prognostic information to ZAP-70-positive patients. Conclusions:,CLLU1 is the first identified CLL specific gene. The CLLU1 mRNA expression level can predict time to initiation of treatment and survival in CLL patients. [source] Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CMLEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2002Yoo-Jin Kim Abstract: The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000,13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000,39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools. [source] Silencing dopamine D3 -receptors in the nucleus accumbens shell in vivo induces changes in cocaine-induced hyperlocomotionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005Amine Bahi Abstract The dopamine D3 receptor (D3R) is an important pharmacotherapeutic target for its potential role in psychiatric disorders and drug dependence. To further explore its function in rats, a regulatable lentivirus, Lenti-D3, holding the rat D3R cDNA, has been constructed as well as three nonregulatable lentiviruses, Lenti-D3-siRNA1, Lenti-D3-siRNA2 and Lenti-D3-siRNA3, expressing small hairpin RNAs, aimed at silencing D3R expression and specifically targeted against different regions of the D3R mRNA. In vitro, Lenti-D3 expressed D3R and could efficiently be blocked with Lenti-D3-Sils. These viruses were stereotaxically injected into the shell part of the nucleus accumbens (NAcc) and effects of passive cocaine delivery on locomotor activity were assessed. Manipulations of D3R levels induced changes in the locomotor stimulant effects of cocaine as compared to control treatment. Suppression of dopamine (DA) D3R in the NAcc by means of local knockdown (with Lenti-D3-Sils) increased locomotor stimulant effects, whereas its overexpression with Lenti-D3 drastically reduced them. The latter effects could be reversed when animals were fed doxycycline, which prevented lentiviral-mediated DA D3R overexpression in the NAcc. Gene expression assessed by quantitative RT-PCR confirmed very efficient gene knockdown in vivo in animals treated with Lenti-D3-Sils (> 93% silencing of D3R gene). Thus D3R expression significantly contributes to behavioural changes associated with chronic cocaine delivery. [source] Expression and regulation of interleukin-10 and interleukin-10 receptor in rat astroglial and microglial cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2002Annemarie Ledeboer Abstract Activated glial cells crucially contribute to brain inflammatory responses. Interleukin-10 (IL-10) is an important modulator of glial cell responses in the brain. In the present study we describe the expression of IL-10 and the IL-10 receptor (IL-10R1) in primary cocultures of rat microglial and astroglial cells. Using quantitative RT-PCR and ELISA, we show that IL-10 mRNA expression and subsequent IL-10 secretion is time-dependently induced by lipopolysaccharide (LPS). IL-10R1, however, is constitutively expressed in glial cell cocultures, as shown by RT-PCR and immunocytochemistry. Radioligand binding studies using 125I-IL-10 reveal that rat glial cells express a single binding site with an apparent affinity of approximately 600 pm for human IL-10. Observations in enriched cultures of either microglial or astroglial cells indicate that both cell types express IL-10 mRNA and are capable of secreting IL-10. Both cell types also express IL-10R1 mRNA and protein. However, in glial cell cocultures immunoreactive IL-10R1 protein is predominantly observed in astrocytes, suggesting that microglial expression of IL-10R1 in cocultures is suppressed by astrocytes. In addition, exogenous IL-10 is highly potent in down-regulating LPS-induced IL-1, and IL-10 mRNA, and, at a higher dose, IL-10R1 mRNA in untreated and LPS-treated cultures, suggesting that IL-10 autoregulates its expression and inhibits that of IL-1, at the transcriptional level. Together the findings support the concept that IL-10, produced by activated microglial and astroglial cells, modulates glia-mediated inflammatory responses through high-affinity IL-10 receptors via paracrine and autocrine interactions. [source] Neuronal nitric oxide synthase (nNOS) mRNA is down-regulated, and constitutive NOS enzymatic activity decreased, in thoracic dorsal root ganglia and spinal cord of the rat by a substance P N-terminal metaboliteEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2001Katalin J. Kovacs Abstract Nitric oxide (NO) in the spinal cord plays a role in sensory and autonomic activity. Pain induced by acetic acid in the abdominal stretch (writhing) assay and hyperalgesia associated with chronic pain are highly sensitive to NO synthase (NOS) inhibitors. Because substance P (SP) is released and up-regulated in some models of chronic pain, we hypothesized that an accumulation of SP metabolites may influence NOS expression and activity. To test this hypothesis, we examined the effect of intrathecally (i.t.) injected substance P (1-7) [SP(1-7)], the major metabolite of SP in the rat, on neuronal NOS (nNOS) mRNA in the thoracic and lumbar spinal cord, dorsal root ganglia (DRG) and on the corresponding constitutive NOS (cNOS) enzyme activity. Detected using quantitative RT-PCR, nNOS mRNA content in the thoracic spinal cord was decreased 6 h after injection of 5 µmol of SP(1-7) and returned to control 2 days later. In thoracic DRG, nNOS mRNA was reduced 48 h after SP(1-7). The cNOS enzymatic activity in thoracic spinal tissue was gradually decreased to a minimum at 72 h. Down-regulation of NOS by SP(1-7) in the thoracic area appears to be highly associated with capsaicin-sensitive primary afferent neurons. No similar changes in either parameter were measured in the lumbar area after SP(1-7). These data suggest that N-terminal SP fragments, which are known to cause long-term antinociception in the writhing assay, may do so by their ability to down-regulate NO synthesis along nociceptive pathways. [source] Identification of novel genes regulated by ,-melanocyte-stimulating hormone in murine bone marrow-derived dendritic cellsEXPERIMENTAL DERMATOLOGY, Issue 9 2004T. Brzoska Many strains of evidence indicate that ,-melanocyte-stimulating hormone (,-MSH) elicits its immunomodulatory activity via binding to melanocortin receptors (MC-Rs) expressed on monocytes and dendritic cells. In order to identify novel target genes regulated by ,-MSH in these cells, we prepared bone marrow-derived dendritic cell precursors from BALB/c mice and treated them with GM-CSF and IL-4 for 6 days. The MC-R profile on these immature dendritic cells was first determined by quantitative RT-PCR. Both transcripts for MC-1R and MC-5R were detected in these cells. Cells were subsequently stimulated with dinitrobenzene sulfonic acid (DNBS), ,-MSH or both substances for 2 or 16 h. After RNA preparation, cDNA synthesis and in vitro transcripton hybridization of biotinylated cRNA samples was performed on MG U74A Affymetrix gene chips. Data evaluation, cleansing, extraction and analysis of the more than 12 000 cloned genes and expressed sequence tags were performed using the GENE DATA ANALYST vs. 1 Expressionist software. Filter criteria included a minimum threshold of 100, normalization by the logarithmic mean and a quality setting of P < 0.04. Changes with a change factor of >2 were regarded as significant. As expected, stimulation with DNBS resulted in induction or upregulation of genes encoding proinflammatory cytokines, growth factors, signal transduction intermediates and transcription factors. Treatment with ,-MSH blocked the DNBS-driven upregulation of several known genes such as IL-1 or CD86. On the other hand, ,-MSH modulated the expression of several novel genes implicated in immunomodulation, e.g. IL-1, converting enzyme, IFN-, receptor, FK506-binding proteins or several neuropeptides and their receptors. These data indicate novel molecular targets by which ,-MSH exerts its immunomodulatory activities in immunocompetent cells. [source] Characterization of chitinase-like proteins (Cg -Clp1 and Cg -Clp2) involved in immune defence of the mollusc Crassostrea gigasFEBS JOURNAL, Issue 14 2007Fabien Badariotti Chitinase-like proteins have been identified in insects and mammals as nonenzymatic members of the glycoside hydrolase family 18. Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes. However, the precise physiological roles of Cg -Clp1 in oysters remain unknown. Here, we report the cloning and the characterization of a new chitinase-like protein (Cg -Clp2) from the oyster Crassostrea gigas. Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling. Both Cg -Clp1- and Cg -Clp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity. [source] Clock-dependent and independent transcriptional control of the two isoforms from the mouse Ror,geneGENES TO CELLS, Issue 12 2008Valérie Mongrain Accumulating evidence indicate that molecular mechanisms generating circadian rhythms display some degree of tissue-specificity. More specifically, distinct patterns of expression for nuclear receptors of the ROR family indicate that the transcriptional control of the clock gene Bmal1 differs among tissues. This study aims to investigate the expression of Ror,isoforms (Ror, and Ror,t) and characterize the molecular mechanisms underlying their tissue-specific expression. The expression of Ror, isoforms was assessed in mouse liver, muscle, thymus and testis throughout 24 h using quantitative RT-PCR. Although the expression of Ror, was rhythmic in the liver and thymus, it was constitutively expressed in muscle and testis. In contrast, the expression of Ror,t was constitutive in all four tissues. Furthermore, rhythmic expression of Ror, was impaired in Clock mutant mice whereas the mutation had no effect on Ror,t expression. In line with these findings, luciferase assays revealed that transcription of the Ror, promoter is clock-controlled whereas that of Ror,t promoter is essentially clock-independent. Our results provide insights into the molecular mechanisms that lead to differential expression of Ror, and Ror,t and are suggestive of a framework that might account for tissue-specific circadian regulation. [source] TNFAIP3 is the target gene of chromosome band 6q23.3-q24.1 loss in ocular adnexal marginal zone B cell lymphomaGENES, CHROMOSOMES AND CANCER, Issue 1 2008Keiichiro Honma The genomic aberrations in extra nodal marginal zone B cell lymphoma vary according to their anatomical origin. This polarization is a reflection of the participation of different genes in the lymphomagenesis of marginal zone B cell lymphoma. We previously demonstrated by means of genome-wide array comparative genomic hybridization (CGH) that the genomic profile of ocular adnexal marginal zone B cell lymphoma is distinct from that of pulmonary or nodal marginal zone B cell lymphoma. The novel finding was a recurrent deletion of a 2.9-Mb region at chromosome band 6q23.3-q24.1, including homozygous loss, in ocular adnexal marginal zone B cell lymphoma. For a more detailed examination of the deletions of 6q23.3-24.1, we used contig bacterial artificial chromosome (BAC) array CGH, containing 24 BAC clones covering the 2.9-Mb region, to analyze nine cases with 6q23.3-q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR. Correlation between genomic loss and expression level was found only for TNFAIP3, demonstrating that TNFAIP3 is a target gene of 6q deletion in ocular adnexal marginal zone B cell lymphoma. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may play an important role in lymphomagenesis and suggests that TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source] Mutations in TREM2 lead to pure early-onset dementia without bone cysts,HUMAN MUTATION, Issue 9 2008Eliane Chouery Abstract A genome-wide screen using 382 STR markers to localize and identify the gene implicated in early-onset dementia (EOD) without bone cysts in a Lebanese family with three affected subjects was conducted. A unique locus homozygous by descent at chromosome 6p21.2 locus was identified. Candidate genes were explored by fluorescent sequencing and the effect of the identified mutation was confirmed by qualitative and quantitative RT-PCR. The genetic analysis revealed a novel deletion, c.40+3delAGG, in the 5' consensus donor splice site in intron 1 of TREM2 gene which is known to be responsible for PLOSL (Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy) also designated as Nasu-Hakola disease. In silico analysis predicted a lower strength for the novel donor splice site. Qualitative RT-PCR revealed normal transcript while quantitative RT-PCR showed over twofold down-regulation of TREM2 transcripts. The expression profile of six genes SPP1, NEDD9, FSCN, BCL3, NFKBIA and CCL2 known as disrupted in TREM2-deficient samples was studied and showed same expression profile as TREM2-mutated samples except for CCL2 which was normally regulated. The significantly-reduced expression of TREM2 in our patients and the expression profiles of the six studied genes confirm a role for TREM2 in this distinct phenotype of EOD without bone cysts. To our knowledge, this is the first report of mutations in TREM2 causing a pure dementia. © 2008 Wiley-Liss, Inc. [source] Immune-privileged embryonic Swiss mouse STO and STO cell-derived progenitor cells: major histocompatibility complex and cell differentiation antigen expression patterns resemble those of human embryonic stem cell linesIMMUNOLOGY, Issue 1 2006Katherine S. Koch Summary Embryonic mouse STO (S, SIM; T, 6-thioguanine resistant; O, ouabain resistant) and 3(8)21-enhanced green fluorescent protein (EGFP) cell lines exhibit long-term survival and hepatic progenitor cell behaviour after xenogeneic engraftment in non-immunosuppressed inbred rats, and were previously designated major histocompatibility complex (MHC) class I- and class II-negative lines. To determine the molecular basis for undetectable MHC determinants, the expression and haplotype of H-2K, H-2D, H-2L and I-A proteins were reassessed by reverse transcriptase,polymerase chain reaction (RT-PCR), cDNA sequencing, RNA hybridization, immunoblotting, quantitative RT-PCR (QPCR), immunocytochemistry and flow cytometry. To detect cell differentiation (CD) surface antigens characteristic of stem cells, apoptotic regulation or adaptive immunity that might facilitate progenitor cell status or immune privilege, flow cytometry was also used to screen untreated and cytokine [interferon (IFN)-,]-treated cultures. Despite prior PCR genotyping analyses suggestive of H-2q haplotypes in STO, 3(8)21-EGFP and parental 3(8)21 cells, all three lines expressed H-2K cDNA sequences identical to those of d-haplotype BALB/c mice, as well as constitutive and cytokine-inducible H-2Kd determinants. In contrast, apart from H-2Ld[LOW] display in 3(8)21 cells, H-2Dd, H-2Ld and I-Ad determinants were undetectable. All three lines expressed constitutive and cytokine-inducible CD34; however, except for inducible CD117[LOW] expression in 3(8)21 cells, no expression of CD45, CD117, CD62L, CD80, CD86, CD90·1 or CD95L/CD178 was observed. Constitutive and cytokine-inducible CD95[LOW] expression was detected in STO and 3(8)21 cells, but not in 3(8)21-EGFP cells. MHC (class I+[LOW]/class II,) and CD (CD34+/CD80,/CD86,/CD95L,) expression patterns in STO and STO cell-derived progenitor cells resemble patterns reported for human embryonic stem cell lines. Whether these patterns reflect associations with mechanisms that are regulatory of immune privilege or functional tissue-specific plasticity is unknown. [source] | |||||||||||||||||||||||||||||||||||||