Improved Transient Protein Expression by pFluNS1 Plasmid

Influenza virus nonstructural protein-1 (NS1) is abundantly expressed in influenza virus infected cells. NS1 is well recognized for counteracting host antiviral activities and regulating host and viral protein expression. When used as a plasmid component in DNA transfection, NS1 was shown to significantly increase expression levels of a cotransfected gene of different plasmid. Our previous studies demonstrated that addition of an NS1 plasmid increased the expression levels of influenza virus secreted neuraminidase (sNA) gene in 293T cells. In this study, we improved the utilization of NS1 as an enhancer for transient protein expression by generating pFluNS1 plasmid to contain two expression cassettes; one encoding an NS1 gene and another encoding a gene of interest. pFluNS1 is expected to codeliver the NS1 gene into the same cells receiving the gene of interest. The plasmid is therefore designed to induce higher protein expression levels than a cotransfection of an NS1 plasmid and a plasmid containing a gene of interest. To test the efficiency of pFluNS1, influenza virus sNA and non-viral DsRed genes were cloned into pFluNS1. The expression of these genes from pFluNS1 was then compared to the expression from a cotransfection of an NS1 plasmid and an expression plasmid coding for sNA or DsRed. We found that gene expression from pFluNS1 reached equal or higher levels to those derived from the cotransfection. Because the expression from pFluNS1 needs only one plasmid, a lesser amount of transfection reagent was required. Thus, the use of pFluNS1 provides a transfection approach that reduces the cost of protein expression without compromising high levels of protein expression. Together, these data suggest that pFluNS1 can serve as a novel alternative for an efficient transient protein expression in mammalian cells.     

p53 protein expression levels as bioindicator of individual exposure to ionizing radiation by flow cytometry

Ionizing radiation (IR) can cause various lesions in DNA, which induce the increase of p53 expression levels in order to repair radiation induced damage. Thus, the correlation between the increase of p53 expression and an irradiation may constitute a fast and powerful method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the aim of this research was to evaluate changes in lymphocyte p53 expression levels, based on flow cytometry, after in vitro irradiation of peripheral blood samples. For the measurement of such expression levels of p53 protein, an investigation was carried out in order to establish a methodology of analysis based on flow cytometry. Hence, relationships among levels of expression of p53 protein with the absorbed dose have been verified. The results presented in this report emphasized flow cytometry as an important tool for the fast evaluation of p53 protein expression levels as bioindicator of individual exposure to acute ionizing radiation.     

Brain HSP70 mRNA expression is linked with plasma cortisol levels in goldfish (Carassius auratus) exposed to a potential predator

We previously found that when goldfish were exposed to a potential predator, bluegills, the goldfish experienced an increase in HSP70 mRNA expression in the brains and increased plasma cortisol levels. In the present study, we examined the potential causative relationship between HSP70 mRNA expression and plasma cortisol levels. Cortisol agonists (corticotropin releasing factor and cortisol) and antagonists (metyrapone and betamethasone) were used to modulate plasma cortisol levels. HSP70 mRNA expression and plasma cortisol levels were analyzed by Northern blotting and ELISA, respectively. Goldfish treated with the cortisol agonists showed marked increases in plasma cortisol levels and also in brain HSP70 mRNA expression. When goldfish were exposed to bluegills, plasma cortisol levels increased and HSP70 mRNA expression was enhanced after 6 hr. However, pre-treatment with the cortisol antagonists 24 hr prior to the exposure inhibited the enhancement as well as the increase in plasma cortisol levels. These results suggest that plasma cortisol plays a key role in the enhancement of brain HSP70 mRNA expression in goldfish stressed by exposure to bluegills.     

Detecting changes in the relative expression of KRAS2 splice variants using polymerase colonies

Because splice variants of a gene with multiple isoforms give rise to proteins with different functions, it seems plausible that changes in the expression levels of the splice variants could be a contributing factor to disease. In fact, recent examples in the literature clearly illustrate that altered expression levels of splice variants may play an important role in disease. Furthermore, these works demonstrate that changes in expression levels could potentially be used to (1) monitor disease progression, (2) diagnose disease, and/or (3) determine disease state. In this work an immobilized form of PCR, known as polony technology, was adapted to quantify the relative expression levels of splice variants. Specifically, the relative expression levels of the two splice variants of the oncogene K-ras, namely, K-RAS2A and K-RAS2B, were determined using polony technology.     

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.     

Variation in the large-scale organization of gene expression levels in the hippocampus relates to stable epigenetic variability in behavior

Background

Despite sharing the same genes, identical twins demonstrate substantial variability in behavioral traits and in their risk for disease. Epigenetic factors–DNA and chromatin modifications that affect levels of gene expression without affecting the DNA sequence–are thought to be important in establishing this variability. Epigenetically-mediated differences in the levels of gene expression that are associated with individual variability traditionally are thought to occur only in a gene-specific manner. We challenge this idea by exploring the large-scale organizational patterns of gene expression in an epigenetic model of behavioral variability.

Methodology/Findings

To study the effects of epigenetic influences on behavioral variability, we examine gene expression in genetically identical mice. Using a novel approach to microarray analysis, we show that variability in the large-scale organization of gene expression levels, rather than differences in the expression levels of specific genes, is associated with individual differences in behavior. Specifically, increased activity in the open field is associated with increased variance of log-transformed measures of gene expression in the hippocampus, a brain region involved in open field activity. Early life experience that increases adult activity in the open field also similarly modifies the variance of gene expression levels. The same association of the variance of gene expression levels with behavioral variability is found with levels of gene expression in the hippocampus of genetically heterogeneous outbred populations of mice, suggesting that variation in the large-scale organization of gene expression levels may also be relevant to phenotypic differences in outbred populations such as humans. We find that the increased variance in gene expression levels is attributable to an increasing separation of several large, log-normally distributed families of gene expression levels. We also show that the presence of these multiple log-normal distributions of gene expression levels is a universal characteristic of gene expression in eurkaryotes. We use data from the MicroArray Quality Control Project (MAQC) to demonstrate that our method is robust and that it reliably detects biological differences in the large-scale organization of gene expression levels.

Conclusions

Our results contrast with the traditional belief that epigenetic effects on gene expression occur only at the level of specific genes and suggest instead that the large-scale organization of gene expression levels provides important insights into the relationship of gene expression with behavioral variability. Understanding the epigenetic, genetic, and environmental factors that regulate the large-scale organization of gene expression levels, and how changes in this large-scale organization influences brain development and behavior will be a major future challenge in the field of behavioral genomics.     

Role of microRNA-21 and microRNA-155 as biomarkers for bronchial asthma

MicroRNA (miRNA)-21 and miRNA-155 are important regulators of gene expression of different immunological molecules. This study aimed to investigate the role of miRNA-21 and miRNA-155 as biomarkers in asthma by comparing their serum expression levels in asthmatic patients to those in healthy controls and correlating their levels with serum IL-4. The expression levels of miRNA-21 and miRNA-155 were evaluated by quantitative RT-PCR. Serum levels of IL-4 were determined using ELISA. Asthmatic patients showed significantly higher serum miRNA-21 and miRNA-155 expression levels compared to controls. A statistically significant positive correlation between the expression levels of miRNA-21 and IL-4 serum levels in asthmatic patients was detected. Nonetheless, no correlation was detected between miRNA-155 expression and each of IL-4 and miRNA-21. A receiver operating characteristic curve analysis showed that at a cut-off value of 1.37, the sensitivity of miRNA-21 as an asthma biomarker was 100% and the specificity was 95%. At a cut-off value of 1.96, the sensitivity of miRNA-155 as an asthma biomarker was 100% and the specificity was 100%. It can be concluded that miRNA-21 and miRNA-155 are potential non-invasive biomarkers in the diagnosis of eosinophilic asthma and its response to therapy.     

Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels

In order to avoid the metabolic burden of protein expression during cell growth, and to avoid potential toxicity of recombinant proteins, microbial expression systems typically utilize regulated expression vectors. In contrast, constitutive expression vectors have usually been utilized for isolation of protein expressing mammalian cell lines. In mammalian systems, inducible expression vectors are typically utilized for only those proteins that are toxic when overexpressed. We developed a tetracycline regulated expression system in CHO cells, and show that cell pools selected in the uninduced state recover faster than those selected in the induced state even though the proteins showed no apparent toxicity or expression instability. Furthermore, cell pools selected in the uninduced state had higher expression levels when protein expression was turned on only in production cultures compared to pools that were selected and maintained in the induced state through production. We show a titer improvement of greater than twofold for an Fc-fusion protein and greater than 50% improvement for a recombinant antibody. The improvement is primarily due to an increase in specific productivity. Recombinant protein mRNA levels correlate strongly with protein expression levels and are highest in those cultures selected in the uninduced state and only induced during production. These data are consistent with a model where CHO cell lines with constitutive expression select for subclones with lower expression levels.     

Insulin stimulation of gene expression mediated by p21ras activation.

In fibroblasts, insulin is a weak mitogen and does not induce expression of c-fos, c-jun or p33. However, increasing the expression levels of either normal p21Hras or the insulin receptor, but not mutant p21Hras, enables insulin to induce the expression of these genes. In cells expressing elevated levels of insulin receptor, this process involves a rapid increase in p21rasGTP levels (from 20% to 70% GTP as a percentage of total guanine nucleotides). No increase in p21rasGTP levels was observed after PDGF and EGF stimulation of cells expressing high levels of the cognate receptor, stressing the specificity of the insulin-induced increase. We conclude that in fibroblasts, p21ras is an intermediate of the insulin signal transduction pathway involved in the regulation of gene expression and mitogenicity.     

Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells.

In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.     

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.     

Variables Affecting In Vivo Performance of High-Capacity Adenovirus Vectors

In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.     

Differential expression of granulysin and perforin by NK cells in cancer patients and correlation of impaired granulysin expression with progression of cancer

Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3(-) CD16(+) cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression ( P<0.005), despite having equally high levels of perforin expression in comparison with healthy controls. The tumor-free patients expressed granulysin at levels similar to healthy controls, while the progressive tumor-bearing patients expressed remarkably lower levels of granulysin compared to healthy controls ( P<0.0001). Similarly, patients with an advanced performance status had significantly fewer granulysin-positive NK cells than healthy controls. Meanwhile, a considerable number of the tumor-bearing patients showed a decrease in the number of circulating NK cells, and a correlation between impaired granulysin expression and reduced circulating NK cells was observed. These findings suggest that the tumor-bearing patients with impaired granulysin expression were in an immunosuppressive state. In conclusion, impaired expression of granulysin by NK cells correlates with progression of cancer, and determination of granulysin expression might prove informative for assessing the immunological condition of cancer patients.     

Evaluation of experimental designs for two-color cDNA microarrays.

The major goal of two-color cDNA microarray experiments is to measure the relative gene expression level (i.e., relative amount of mRNA) of each gene between samples in studies of gene expression. More specifically, given an N-sample experiment, we need all N(N - 1)/2 relative expression levels of all sample pairs of each gene for identification of the differentially expressed genes and for clustering of gene expression patterns. However, the intensities observed from two-color cDNA microarray experiments do not simply represent the relative gene expression level. They are composed of signal (gene expression level), noise, and other factors. In discussions on the experimental design of two-color cDNA microarray experiments, little attention has been given to the fact that different combinations of test and control samples will produce microarray intensities data with varying intrinsic composition of factors. As a consequence, not all experimental designs for two-color cDNA microarray experiments are able to provide all possible relative gene expression levels. This phenomenon has never been addressed. To obtain all possible relative gene expression levels, a novel method for two-color cDNA microarray experimental design evaluation is necessary that will allow the making of an accurate choice. In this study, we propose a model-based approach to illustrate how the factor composition of microarray intensities changed with different experimental designs in two-color cDNA microarray experiments. By analyzing 12 experimental designs (including 5 general forms), we demonstrate that not all experimental designs are able to provide all possible relative gene expression levels due to the differences in factor composition. Our results indicate that whether an experimental design can provide all possible relative expression levels of all sample pairs for each gene should be the first criterion to be considered in an evaluation of experimental designs for two-color cDNA microarray experiments.     

Development of tyrosine aminotransferase in perinatal rat liver: changes in functional messenger RNA and the role of inducing hormones

Expression of the hepatic enzyme tyrosine aminotransferase was analyzed in the perinatal period of development in the rat, when this expression undergoes significant changes associated with hepatocyte differentiation. In late prenatal liver both enzyme and functional mRNA gene products are present at levels 10- to 15-fold below those in the fully differentiated adult liver. This low level of expression in fetal liver is refractory to induction by glucocorticoids, but both gene products are increased to a limited extent by cyclic AMP. This induction by cyclic AMP (cAMP) does not confer glucocorticoid-responsiveness on expression. By 3 hr after birth both functional mRNA and enzyme levels are significantly increased, an increase which continues until a peak is reached at 12 hr that is appreciably above the adult levels. Both gene products then decline until adult levels are reached by 24 hr. The postnatal shift in aminotransferase expression is accompanied by acquisition of the capacity to respond to glucocorticoids. Treatment of newborns with an antiglucocorticoid steroid or with glucose suppresses the postnatal overshoot of expression, but neither treatment affects the increase from fetal to adult levels of expression. The results indicate that prior to birth, expression of the aminotransferase gene is partially repressed, a repression that is lifted essentially immediately upon birth. The hormones capable of inducing aminotransferase synthesis have no apparent necessary role in this process.