Validation of reference genes for quantitative real-time polymerase chain reaction assay of honeybee under various pesticide treatment conditions

In quantitative real-time polymerase chain reaction (qRT-PCR), target gene expression levels are normalized to internal reference gene(s) that are stably expressed across different conditions to determine whether they are up- or down-regulated. Therefore, it is essential to select appropriate reference gene(s) for the accurate comparison of target gene expression across different experimental conditions. Honeybee colonies can be damaged due to pesticide exposure, resulting in a decline of their population. Determination of gene expression levels is important for understanding the physiological response of honeybees to pesticide exposure. Therefore, in this study, we used qRT-PCR to analyze the expression stability of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAB1a) in honeybees subjected to treatment with different dosages and exposure durations of seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin) using four programs (geNorm, NormFinder, BestKeeper, and RefFinder). Subsequently, the expression levels of the target genes (PER, FOR, and EGR1) were calculated using different normalization methods and compared. Based on our collective results, we propose RPS5 as the most appropriate reference gene for the normalization of target gene expression levels in qRT-PCR assays for honeybees under various conditions of pesticide exposure, including pesticide type, exposure duration, and concentration.     

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA

Background  

The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees.     

Computational expression deconvolution in a complex mammalian organ

Background  

Microarray expression profiling has been widely used to identify differentially expressed genes in complex cellular systems. However, while such methods can be used to directly infer intracellular regulation within homogeneous cell populations, interpretation of in vivo gene expression data derived from complex organs composed of multiple cell types is more problematic. Specifically, observed changes in gene expression may be due either to changes in gene regulation within a given cell type or to changes in the relative abundance of expressing cell types. Consequently, bona fide changes in intrinsic gene regulation may be either mimicked or masked by changes in the relative proportion of different cell types. To date, few analytical approaches have addressed this problem.     

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.     

Over expression of the selectable marker blasticidin S deaminase gene is toxic to human keratinocytes and murine BALB/MK cells

Background  

The blasticidin S resistance gene (bsr) is a selectable marker used for gene transfer experiments. The bsr gene encodes for blasticidin S (BS) deaminase, which has a specific activity upon BS. Therefore, its expression is supposed to be harmless in cells. The work reported on herein consisted of experiments to verify a possible toxicity of bsr on mammalian cells, which include several cell lines and primary cultures.     

Long-term maintenance of in vitro cultured honeybee (Apis mellifera) embryonic cells

Background  

In vitro cultivation of cells allows novel investigation of in vivo- mechanisms and is a helpful tool in developmental biology, biochemistry and functional genomics. Numerous cell lines of insect species, e.g., silkworm and mosquito, have been reported. However, this is not the case for successful long-term cultivation of cells in honeybees.     

Industrial-scale production and purification of a heterologous protein in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> using the nisin-controlled gene expression system NICE: The case of lysostaphin

Background  

The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation.     

Subinhibitory concentrations of antibiotics affect stress and virulence gene expression in Listeria monocytogenes and cause enhanced stress sensitivity but do not affect Caco‐2 cell invasion

Aims

Antibiotics can act as signal molecules and affect bacterial gene expression, physiology and virulence. The purpose of this study was to determine whether subinhibitory antibiotic concentrations alter gene expression and physiology of Listeria monocytogenes.

Methods and Results

Using an agar‐based screening assay with promoter fusions, 14 of 16 antibiotics induced or repressed expression of one or more stress and/or virulence genes. Despite ampicillin‐induced up‐regulation of PinlA‐lacZ expression, Caco‐2 cell invasion was not affected. Subinhibitory concentrations of ampicillin and tetracycline caused up‐ and down‐regulation of stress response genes, respectively, but both antibiotics caused increased sensitivity to acid stress. Six combinations of gene‐antibiotic were quantified in broth cultures and five of the six resulted in the same expression pattern as the agar‐based assay.

Conclusions

Antibiotics affect virulence and/or stress gene expression; however, altered expression could not predict changes in phenotypic behaviour. Subinhibitory concentrations of antibiotics led to increased acid sensitivity, and we speculate that this is attributed to changes in cell envelope or reduced σ^B‐dependent gene expression.

Significance and Impact of the Study

Although subinhibitory concentrations of antibiotics affect gene expression in L. monocytogenes, the changes did not increase virulence but did enhance the acid sensitivity.     

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

Background  

Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae.     

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus

Background  

Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.     

The Virtual Insect Brain protocol: creating and comparing standardized neuroanatomy

Background  

In the fly Drosophila melanogaster, new genetic, physiological, molecular and behavioral techniques for the functional analysis of the brain are rapidly accumulating. These diverse investigations on the function of the insect brain use gene expression patterns that can be visualized and provide the means for manipulating groups of neurons as a common ground. To take advantage of these patterns one needs to know their typical anatomy.     

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

Background  

Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken.     

Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells

Background

Several approaches for gene therapy of cystic fibrosis using viral and non‐viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO‐K1 cells, since these cells have been extensively used for heterologous CFTR expression.

Methods

An established in vitro gene transfer system based on antibiotic‐mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole‐cell patch‐clamp recordings.

Results

L. monocytogenes mediated gene transfer to CHO‐K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO‐K1 cells.

Conclusions

This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

Background  

PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.