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Results for “Gene Expression”

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At least 109 records · Page 6Linked to original sources

PubMed2000

Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression.

Gene transfer into muscle by electroporation with low-voltage and long-pulse (LV/LP, 100 V/50 msec) currents was shown to be more efficient than simple intramuscular DNA injection. Nevertheless, transgene expression declined from day 7 and only reached 10% of the maximum 3 weeks after electroporation. We have optimized electroporation conditions including voltage, pulse number, and the amount of injected luciferase-encoding plasmid DNA in the tibialis anterior muscle. Using high-voltage and short-pulse (HV/SP, 900 V/100 microsec) currents, we observed an average 500-fold increase in luciferase expression, in comparison with nonelectroporated muscle. Moreover, sustained and long-lasting gene expression was observed for at least 6 months. When we compared HV/SP currents with LV/LP currents, luciferase expression was similar 24 hr after electroporation. One month later, whereas luciferase expression was stable in muscle electroporated with HV/SP currents, it decreased 600-fold in muscle electroporated with LV/LP currents. In conclusion, electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression in muscle.

Animals↗

PubMed2000

Characterization of genomically amplified segments using PCR: optimizing relative-PCR for reliable and simple gene expression and gene copy analyses.

Gene amplification is one of the mechanisms for oncogene activation in solid tumors. The size of the amplified regions may vary considerably among individual tumors, and more than one gene may be affected within the same amplicon. The main objective in analyzing genomic amplifications has therefore been to map the shortest region involved and to identify genes with increased expression as a result of the increased gene copy number. To facilitate such an analysis, we have developed simple polymerase chain reaction (PCR) procedures using the internal standards beta-actin (ACTB) and L1Hs for gene expression and gene copy number analyses, respectively. We used cDNA derived from pancreatic carcinoma cell lines, and genomic DNA extracted from the same cell lines, as templates in the gene expression and in the gene copy number analyses, respectively. To determine the optimal number of PCR cycles, dilution series of the templates were made. Furthermore, competing primers were used to adjust for differences in target sequence levels. We show that by these simple means it is possible to determine optimal conditions for expression analyses. In addition, the procedure was adapted for the analysis of gene copy number changes at the genomic level using L1Hs as the internal standard. This PCR method makes it possible to produce detailed gene copy number profiles of amplified genomic regions.

DNA Primers↗

PubMed2005

Scintigraphic imaging of gene expression and gene transfer.

Assessment of gene function following the completion of human genome sequencing can be performed with nuclear medicine procedures. These techniques may be applied for the determination of gene function and regulation using established and new tracers or using in vivo reporter genes such as genes encoding enzymes, receptors, antigens, or transporters. Visualization of in vivo reporter gene expression can be done using radiolabeled substrates, antibodies, or ligands. Combinations of specific promoters and in vivo reporter genes may deliver information about the regulation of the corresponding genes. The role of radiolabeled antisense molecules for the analysis of mRNA content has to be investigated. However, possible applications are therapeutic interventions using triplex oligonucleotides with therapeutic isotopes, which can be brought near to specific DNA sequences to induce DNA strand breaks at selected loci. Imaging of labeled siRNA's makes sense if these are used for therapeutic purposes in order to assess the delivery of these new drugs to their target tissue. Pharmacogenomics will identify new surrogate markers for therapy monitoring which may represent potential new tracers for imaging.

Animals↗

PubMed2005

Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors.

UNLABELLED: Indirect, noninvasive imaging of therapeutic gene expression based on levels of reporter gene expression is a powerful tool to devise improved therapeutic strategies in cancer gene therapy. The use of bicistronic vectors carrying internal ribosome entry sites (IRESs) allows the coexpression of multiple gene products from the same promoter but leads to considerable attenuation of the downstream gene. In this study, we describe the use of 10 linked copies of the Gtx (homeodomain protein) IRES (abbreviated as SIRES) in place of the encephalomyocarditis (EMCV) IRES in mediating downstream reporter gene expression in cell culture and in vivo. METHODS: We constructed several plasmid vectors carrying different upstream and downstream reporter genes (herpes simplex virus type I thymidine kinase [tk], firefly luciferase [fl], and Renilla luciferase [rl]) placed between EMCV IRES and SIRES segments. RL, FL, and TK enzyme activities in N2a, C6, and 293 cells transiently transfected with these vectors were found to be significantly higher for the SIRES vectors than for the EMCV IRES vectors. For in vivo experiments, 4 stably transfected N2a cell lines were implanted in nude mice. The mice were imaged for rl and fl gene expression using a charged-coupled device (CCD) camera. For bioluminescence and microPET imaging of downstream gene expression of fl and tk genes, respectively, mice carrying 4 stably transfected xenografts were imaged using the CCD camera and microPET. RESULTS: In cell culture, using rl as the upstream gene, we demonstrate that the expression of the downstream tk gene is 12-fold greater using SIRES when compared with EMCV IRES. Furthermore, the expression of the 2 genes was highly correlated in N2a cells. In vivo bioluminescence imaging using 4 stably transfected N2a cell lines revealed increasing levels of rl and fl gene expression. Bioluminescence and microPET, respectively, of fl and tk reporter gene expression in nude mice bearing N2a tumor xenografts showed the gene expression mediated by SIRES to be 4- and 8-fold higher, respectively, than EMCV IRES. CONCLUSION: These findings support the use of SIRES bicistronic vectors for a better assessment of therapeutic gene expression based on reporter gene expression in living subjects.

Animals↗

PubMed2000

Genes and gene expression.

Gene therapy is the process whereby a therapeutic protein is synthesized from a DNA molecule (gene) that has been inserted into the cells. The goal is to produce the desired protein in the proper quantity in the proper location. Successful designing of vectors for gene therapy requires knowledge of gene structure and regulation. The gene is comprised of protein-coding sequences (called exons) that are interrupted by noncoding sequences (called introns). The expression of a gene in a tissue is regulated by proteins that bind to specific deoxyribonucleic acid sequences generally upstream from the first exon, in the deoxyribonucleic acid promoter domain. Understanding the mechanisms of gene regulation provides the deoxyribonucleic acid sequences necessary to direct expression of specific genes in the right tissue at the proper time in the desired amount.

Animals↗

PubMed2006

Meningococcal adhesion suppresses proapoptotic gene expression and promotes expression of genes supporting early embryonic and cytoprotective signaling of human endothelial cells.

Neisseria meningitidis colonizes the human nasopharynx and occasionally causes lethal or damaging septicemia and meningitis. Here, we examined the adherence-mediated signaling of meningococci to human cells by comparing gene expression profiles of human umbilical vein endothelial cells (HUVEC) infected by adherent wild-type, frpC-deficient mutant, or the nonadherent (DeltapilD) N. meningitidis. Pili-mediated adhesion of meningococci resulted in alterations of expression levels of human genes known to regulate apoptosis, cell proliferation, inflammatory response, adhesion and genes for signaling pathway proteins such as TGF-beta/Smad, Wnt/beta-catenin and Notch/Jagged. This reveals that adhering piliated meningocci manipulate host signaling pathways controlling cell proliferation while establishing a commensal relationship.

Apoptosis↗

PubMed1989

Codon contexts from weakly expressed genes reduce expression in vivo.

Nucleotides that neighbor codons in Escherichia coli genes are highly non-random. Furthermore, these context biases are stronger and extend farther from the codon in weakly expressed than in highly expressed genes. We therefore suggested that codon contexts are selected to reduce gene expression levels. We now compare the expression levels of lacZ genes containing two specific coding sequences (context inserts). One context insert represents contexts seen in weakly expressed genes (low variant); the other represents contexts seen in highly expressed genes (high variant). The two variants have identical nucleotide and codon compositions, and encode the same protein. A permutation of four nucleotides, which changes eight codon:codon interfaces of 1043, comprises the only difference between the high and low context variant genes. In three different lacZ mRNAs, the low variant was expressed at a level significantly below that of the high variant. This context effect depends entirely on translation of the contexts in the correct frame; its magnitude depends in part on the placement of other features (e.g. transcriptional pauses and terminators, or perhaps other slow codons or contexts) in the mRNAs. Changing the ribosome density on the message by changing the ribosome binding site distinguishes between dropoff, interference and polarity, three fundamentally different types of models for the context effect. The expression difference between context variants is eliminated by both increases and decreases in the ribosome initiation frequency, as uniquely predicted by the polarity model. In fact, data from all constructions are accommodated by a model in which slow translation of the low context insert increases rho-dependent transcriptional termination within the test gene. The data suggest that the rates of translational initiation and elongation are poised with respect to the rate of transcriptional elongation so that all are influential in setting the expression level of wild-type lacZ. We conclude that context-induced polarity will exist in genes wherever low and reproducible gene product levels have been selected.

Amino Acid Sequence↗

PubMed2001

Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in gram-negative bacteria.

The construction of several stable RK2-derived cloning vectors for the analysis of gene expression and function in gram-negative bacteria is reported. Plasmid stability is conferred by the RK2 par locus or by insertion of the spsAB or spsCD symbiotic plasmid stability loci from pNGR234a of Rhizobium sp. NGR234. The vectors carry multiple cloning sites with protection against read-through transcriptional activity of vector sequences. Vector derivatives with the constitutive nptII promoter or a promoterless gusA gene are suitable for the study of gene function or regulation in bacteria.

Azospirillum brasilense↗

PubMed2003

Identification of epilepsy-related genes by gene expression profiling in the hippocampus of genetically epileptic rat.

Ihara epileptic rat (IER) is an animal model of temporal lobe epilepsy (TLE) with genetically programmed microdysgenesis in the hippocampal formation. The neuronal microdysgenesis is thought to be a cause for recurrent spontaneous seizures. To identify differentially expressed genes in the hippocampus of IER in comparison to control Wistar rat, we performed serial analysis of gene expression (SAGE). As many as 21 differentially expressed genes were identified.

Animals↗

PubMed2002

[Pulmonary emphysema in klotho mutant mice (deficient in klotho gene expression): klotho gene essential in postnatal pulmonary integrity].

The homozygous mutant klotho (KL-/-) mouse, which is deficient in klotho gene expression, exhibits multiple phenotypes resembling human aging that include a short life span, arteriosclerosis, osteoporosis, skin atrophy, and ectopic calcifications. Histologic examination of KL-/- mice at 4 weeks of age reveals pulmonary emphysema. The emphysematous changes are not caused by a developmental defect or hypoplasia of the lung. The lungs of heterozygous mutant klotho (KL+/-) mice show emphysematous changes and alveolar calcification which are almost identical to those in KL-/- mice. These observations indicate that the klotho gene expression is essential to maintaining normal alveolar architecture in postnatal life. Pulmonary function tests reveal prolonged expiration time in KL-/- mice. The expression of type IV collagen and surfactant protein-A mRNA is markedly upregulated in KL-/- mice, suggesting that a defect in matrix synthesis or in type II pneumocyte function, or both, may be involved in the pathogenesis of pulmonary emphysema in this mutant. Several animal strains have been reported to develop pulmonary emphysema, but the klotho mouse is unique because it simultaneously exhibits various aging phenotypes. Analysis of pulmonary emphysema in klotho mice will provide a unique insight into the relationship between pulmonary emphysema and aging.

Aging↗

PubMed1998

Chromatin structure and cardiac gene expression.

Gene activation is often preceded by or accompanied by a perturbation of the chromatin structure. Recently, several co-factors of transcription factors have been identified as histone acetyltransferases. In addition, retinoblastoma protein and some co-factors can form a complex with histone deacetylases. These discoveries provide direct evidence that modification of chromatin structure is crucial for gene activation. However, the role of chromatin structure in cardiac-specific expression has not yet been elucidated. The potential significance of chromatin structure in cardiac-specific gene expression is indicated by: (1) heterogeneous human SWI/SNF chromatin remodelling factors among various tissues; (2) several chromatin remodelling factors shown to be expressed preferentially in the heart; (3) the demonstration of chromatin remodelling of the cardiac beta-myosin heavy chain gene (MyHC) during cardiac development. We therefore propose to study cardiac-specific chromatin remodelling activity in order to elucidate mechanisms controlling the reactivation of the fetal heart genetic program in the hypertrophic heart.

Animals↗

PubMedDate not supplied

Relationship between DNA mismatch repair genes expression, Ku-genes expression and ploidy-related parameters in the progression of pigmented lesions of the skin.

Defects of DNA repair systems in cutaneous tumours are related to DNA mismatch repair genes (MLH1, MSH2, PMS1, PMS2) and Ku70/80 genes involved in double- strand repair. In this study we investigated the statistical relationship between these systems and DNA-ploidy-related parameters in 19 naevus cell naevi, 23 lentigos maligna, 76 primary melanomas and 31 melanoma metastases, applying the correlation coefficient according to Spearman. In naevi significant correlations were found between Ku70/80 gene expression and some ploidy-related parameters. In lentigos, additionally, some significant correlations between the expression of DNA mismatch repair genes were found. Similar results were demonstrated for primary melanomas. In metastases no one significant correlation between DNA mismatch repair genes and Ku-genes was present. We postulate that DNA mismatch repair genes and Ku70/80 genes are functionally independent and that some of them are able to influence ploidy-related parameters.

Adaptor Proteins, Signal Transducing↗

PubMed2002

A regression-based method to identify differentially expressed genes in microarray time course studies and its application in an inducible Huntington's disease transgenic model.

Time-course studies with microarray technologies provide enormous potential for exploring underlying mechanisms of biological phenomena in many areas of biomedical research, but the large amount of gene expression data generated by such studies also presents great challenges to data analysis. Here we introduce a regression-based statistical modeling approach that identifies differentially expressed genes in microarray time-course studies. To illustrate this method, we applied it to data generated from an inducible Huntington's disease transgenic model. The regression method accounts for the induction process, incorporates relevant experimental information, and includes parameters that specifically address the research interest: the temporal differences in gene expression profiles between the mutant and control mice over the time course, in addition to heterogeneities that commonly exist in microarray data. Least-squares and estimating equation techniques were used to estimate parameters and variances, and inferences were made based on efficient and robust Z-statistics under a set of well-defined assumptions. A permutation test was also used to estimate the number of false-positives, providing an alternative measurement of statistical significance useful for investigators to make decisions on follow-up studies.

Animals↗

PubMed2006

Probe-level measurement error improves accuracy in detecting differential gene expression.

MOTIVATION: Finding differentially expressed genes is a fundamental objective of a microarray experiment. Numerous methods have been proposed to perform this task. Existing methods are based on point estimates of gene expression level obtained from each microarray experiment. This approach discards potentially useful information about measurement error that can be obtained from an appropriate probe-level analysis. Probabilistic probe-level models can be used to measure gene expression and also provide a level of uncertainty in this measurement. This probe-level measurement error provides useful information which can help in the identification of differentially expressed genes. RESULTS: We propose a Bayesian method to include probe-level measurement error into the detection of differentially expressed genes from replicated experiments. A variational approximation is used for efficient parameter estimation. We compare this approximation with MAP and MCMC parameter estimation in terms of computational efficiency and accuracy. The method is used to calculate the probability of positive log-ratio (PPLR) of expression levels between conditions. Using the measurements from a recently developed Affymetrix probe-level model, multi-mgMOS, we test PPLR on a spike-in dataset and a mouse time-course dataset. Results show that the inclusion of probe-level measurement error improves accuracy in detecting differential gene expression. AVAILABILITY: The MAP approximation and variational inference described in this paper have been implemented in an R package pplr. The MCMC method is implemented in Matlab. Both software are available from http://umber.sbs.man.ac.uk/resources/puma.

Algorithms↗

PubMed2006

Antigen-induced differential gene expression in lymphocytes and gene expression profile in synovium prior to the onset of arthritis.

To explore early signature genes playing critical roles in the initial steps in an autoimmune murine model of rheumatoid arthritis (RA) (proteoglycan (PG)-induced arthritis; PGIA), we performed gene expression profiling of "arthritogenic" spleen cells stimulated with cartilage PG, and compared them to differentially expressed genes, identified in joints prior to the onset of arthritis, and then in the acute and chronic phases of the disease. A total of 280 genes were up-regulated and 226 genes were suppressed in in vitro PG-stimulated lymphocytes at a minimum of 2-fold expression change. Functional gene classification identified several major clusters of biological activity. Expression of immunoglobulin genes (66 transcripts) was downregulated by approximately 3.7-fold, whereas most of the other genes with immune/inflammation-associated functions such as interleukins (IL-1, -2, -4, -6, -10, -12, -16, -17), chemokine receptors and their ligands (Cxcl1, Ccl2, 7, 8, 9, 10, 22, Ccr2, Ccr5), and major components of the complement cascade were upregulated. Using adoptive disease transfer with stimulated lymphocytes into SCID mice, followed by gene expression profiling of SCID paws, indicated that 37 genes were differentially expressed in yet non-inflamed (pre-arthritic) paws; these genes were related mostly to chemokine, IFN-gamma and TNF-alpha signaling. However, the majority of differentially expressed immune response-related genes were silent in pre-arthritic joints, and only 12 genes were found differentially expressed both in antigen (PG)-stimulated lymphocytes and in the synovium prior to the onset of arthritis. Most of these "arthritis-initiation" genes belonged to chemokine mediated cell motility. Transcripts of chemokine receptor 5 (Ccr5), chemokine ligand 7 (Ccl7) and IFN-gamma-inducible proteins (Ifi47) and GTP-ase 1 were expressed at the highest levels in both antigen-stimulated lymphocytes and pre-inflamed synovium, which suggests a key role of these genes in both lymphocyte maturation and arthritis initiation.

Adoptive Transfer↗

PubMed2002

In vivo analysis of glucocorticoid-induced reporter gene expression using gene gun DNA delivery.

Glucocorticoid regulates various physiological processes via the activation and repression of gene expression. The anti-inflammatory effects and the adverse effects are believed to be dependent on the repression and the activation of genes, respectively. Reporter gene assay is a useful technique to separately evaluate these two functions and has been used for in vitro screening of novel ligands for the glucocorticoid receptor (GR). We report here the application of a reporter gene assay for the in vivo determination of the GR-mediated gene activation. A reporter plasmid containing glucocorticoid response elements was introduced to abdominal mouse skin using a gene gun. Administration of prednisolone induced the expression of the reporter gene, only when the GR expression plasmid was co-transfected with the reporter plasmid. Endogenous levels of corticosterone appeared to be negligible in this protocol. The dose response for this induction was comparable to those for the decreases in thymus weight and serum corticosterone. These results suggest that gene gun-mediated skin transfection enables the in vivo reporter gene assay and that this technique can be used to predict the potency of ligands for the GR-mediated gene activation.

Animals↗

PubMed2006

Baculovirus-mediated gene delivery into Mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression.

Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

Animals↗

PubMed2005

Chromosomal clustering and GATA transcriptional regulation of intestine-expressed genes in C. elegans.

We used mRNA tagging to identify genes expressed in the intestine of C. elegans. Animals expressing an epitope-tagged protein that binds the poly-A tail of mRNAs (FLAG::PAB-1) from an intestine-specific promoter (ges-1) were used to immunoprecipitate FLAG::PAB-1/mRNA complexes from the intestine. A total of 1938 intestine-expressed genes (P<0.001) were identified using DNA microarrays. First, we compared the intestine-expressed genes with those expressed in the muscle and germline, and identified 510 genes enriched in all three tissues and 624 intestine-, 230 muscle- and 1135 germ line-enriched genes. Second, we showed that the 1938 intestine-expressed genes were physically clustered on the chromosomes, suggesting that the order of genes in the genome is influenced by the effect of chromatin domains on gene expression. Furthermore, the commonly expressed genes showed more chromosomal clustering than the tissue-enriched genes, suggesting that chromatin domains may influence housekeeping genes more than tissue-specific genes. Third, in order to gain further insight into the regulation of intestinal gene expression, we searched for regulatory motifs. This analysis found that the promoters of the intestine genes were enriched for the GATA transcription factor consensus binding sequence. We experimentally verified these results by showing that the GATA motif is required in cis and that GATA transcription factors are required in trans for expression of these intestinal genes.

Animals↗