Novel developments for improved detection of specific mRNAs by DNA chips

Microarrays have revolutionized gene expression analysis as they allow for highly parallel monitoring of mRNA levels of thousands of genes in a single experiment. Since their introduction some 15 years ago, substantial progress has been achieved with regard to, e.g., faster or more sensitive analyses. In this review, interesting new approaches for a more sensitive detection of specific mRNAs will be highlighted. Particularly, the potential of electrical DNA chip formats that allow for faster mRNA analyses will be discussed.     

Cowpea mosaic virus nanoscaffold as signal enhancement for DNA microarrays

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.     

Inbreeding effects on gene‐specific DNA methylation among tissues of Chinook salmon

Inbreeding depression is the loss of fitness resulting from the mating of genetically related individuals. Traditionally, the study of inbreeding depression focused on genetic effects, although recent research has identified DNA methylation as also having a role in inbreeding effects. Since inbreeding depression and DNA methylation change with age and environmental stress, DNA methylation is a likely candidate for the regulation of genes associated with inbreeding depression. Here, we use a targeted, multigene approach to assess methylation at 22 growth‐, metabolic‐, immune‐ and stress‐related genes. We developed PCR‐based DNA methylation assays to test the effects of intense inbreeding on intragenic gene‐specific methylation in inbred and outbred Chinook salmon. Inbred fish had altered methylation at three genes, CK‐1, GTIIBS and hsp70, suggesting that methylation changes associated with inbreeding depression are targeted to specific genes and are not whole‐genome effects. While we did not find a significant inbreeding by age interaction, we found that DNA methylation generally increases with age, although methylation decreased with age in five genes, CK‐1, IFN‐?, HNRNPL, hsc71 and FSHb, potentially due to environmental context and sexual maturation. As expected, we found methylation patterns differed among tissue types, highlighting the need for careful selection of target tissue for methylation studies. This study provides insight into the role of epigenetic effects on ageing, environmental response and tissue function in Chinook salmon and shows that methylation is a targeted and regulated cellular process. We provide the first evidence of epigenetically based inbreeding depression in vertebrates.     

Concentration of mammalian genomic DNA using two‐phase aqueous micellar systems

The concentration of biomarkers, such as DNA, prior to a subsequent detection step may facilitate the early detection of cancer, which could significantly increase chances for survival. In this study, the partitioning behavior of mammalian genomic DNA fragments in a two‐phase aqueous micellar system was investigated using both experiment and theory. The micellar system was generated using the nonionic surfactant Triton X‐114 and phosphate‐buffered saline (PBS). Partition coefficients were measured under a variety of conditions and compared with our theoretical predictions. With this comparison, we demonstrated that the partitioning behavior of DNA fragments in this system is primarily driven by repulsive, steric, excluded‐volume interactions that operate between the micelles and the DNA fragments, but is limited by the entrainment of micelle‐poor, DNA‐rich domains in the macroscopic micelle‐rich phase. Furthermore, the volume ratio, that is, the volume of the top, micelle‐poor phase divided by that of the bottom, micelle‐rich phase, was manipulated to concentrate DNA fragments in the top phase. Specifically, by decreasing the volume ratio from 1 to 1/10, we demonstrated proof‐of‐principle that the concentration of DNA fragments in the top phase could be increased two‐ to nine‐fold in a predictive manner. Biotechnol. Bioeng. 2009;102: 1613–1623. © 2008 Wiley Periodicals, Inc.     

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two‐dimensional difference gel electrophoresis (2D‐DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D‐DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D‐DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample. Biotechnol. Bioeng. 2010; 105: 306–316. © 2009 Wiley Periodicals, Inc.     

Identification and characterization of site‐specific N‐glycosylation in the potassium channel Kv3.1b

The potassium ion channel Kv3.1b is a member of a family of voltage‐gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N‐glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)‐glycosylated Kv3.1b reached the plasma membrane, whereas N220‐glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER‐retained Kv3.1b proteins were susceptible to degradation, when co‐expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex₃HexNAc₄Fuc₁ glycan as the major glycan component of the N229‐glycosylated Kv3.1b protein, as opposed to a high‐mannose type Man₈GlcNAc₂ glycan for N220‐glycosylated Kv3.1b. Taken together, these results suggest that trafficking‐dependent roles of the Kv3.1b potassium channel are dependent on N229 site‐specific glycosylation and N‐glycan structure, and operate through a mechanism whereby specific N‐glycan structures regulate cell surface expression.     

A framework for developing and validating taxon‐specific primers for specimen identification from environmental DNA

Taxon‐specific DNA tests are applied to many ecological and management questions, increasingly using environmental DNA (eDNA). eDNA facilitates noninvasive ecological studies but introduces additional risks of bias and error. For effective application, PCR primers must be developed for each taxon and validated in each system. We outline a nine step framework for the development and validation of taxon‐specific primers for eDNA analysis in ecological studies, involving reference database construction, phylogenetic evaluation of the target gene, primer design, primer evaluation in silico, and laboratory evaluation of primer specificity, sensitivity and utility. Our framework makes possible a rigorous evaluation of likely sources of error. The first five steps can be conducted relatively rapidly and (where reference DNA sequences are available) require minimal laboratory resources, enabling assessment of primer suitability before investing in further work. Steps six to eight require more costly laboratory analyses but are essential to evaluate risks of false‐positive and false‐negative results, while step 9 relates to field implementation. As an example, we have developed and evaluated primers to specifically amplify part of the mitochondrial ND2 gene from Australian bandicoots. If adopted during the early stages of primer development, our framework will facilitate large‐scale implementation of well‐designed DNA tests to detect specific wildlife from eDNA samples. This will provide researchers and managers with an understanding of the strengths and limitations of their data and the conclusions that can be drawn from them.     

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l‐methionine‐induced zebrafish with schizophrenia‐like symptoms

Single nucleotide polymorphisms (SNPs) in the human type A gamma‐aminobutyric acid (GABA) receptor β₂ subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l ‐Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET‐induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET‐induced schizophrenia‐like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET‐triggered schizophrenia‐like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T‐maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non‐significant in MET‐triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET‐triggered adult zebrafish with schizophrenia‐like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA_A receptor β₂ subunit involvement in the schizophrenia‐like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research.     

Target‐Activated DNA Polymerase Activity for Sensitive RNase H Activity Assay

Herein, the ribonuclease H (RNase H) activity assay based on the target‐activated DNA polymerase activity is described. In this method, a detection probe composed of two functional sequences, a binding site for DNA polymerase and a catalytic substrate for RNase H, serves as a key component. The detection probe, at its initial state, suppresses the DNA polymerase activity, but it becomes destabilized by RNase H, which specifically hydrolyzes RNA in RNA/DNA hybrid duplexes. As a result, DNA polymerase recovers its activity and initiates multiple primer extension reactions in a separate TaqMan probe‐based signal transduction module, leading to a significantly enhanced fluorescence “turn‐on” signal. This assay can detect RNase H activity as low as 0.016 U mL^?1 under optimized conditions. Furthermore, its potential use for evaluating RNase H inhibitors, which have been considered potential therapeutic agents against acquired immune deficiency syndrome (AIDS), is successfully explored. In summary, this approach is quite promising for the sensitive and accurate determination of enzyme activity and inhibitor screening.     

A novel regulatory element (E77) isolated from CHO‐K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO‐K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO‐K1 genomic DNA fragments with a CMV promoter‐driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study.     

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

Methyl CpG Binding Domain Ultra‐Sequencing: a novel method for identifying inter‐individual and cell‐type‐specific variation in DNA methylation

Experience‐dependent changes in DNA methylation can exert profound effects on neuronal function and behaviour. A single learning event can induce a variety of DNA modifications within the neuronal genome, some of which may be common to all individuals experiencing the event, whereas others may occur in a subset of individuals. Variations in experience‐induced DNA methylation may subsequently confer increased vulnerability or resilience to the development of neuropsychiatric disorders. However, the detection of experience‐dependent changes in DNA methylation in the brain has been hindered by the interrogation of heterogeneous cell populations, regional differences in epigenetic states and the use of pooled tissue obtained from multiple individuals. Methyl CpG Binding Domain Ultra‐Sequencing (MBD Ultra‐Seq) overcomes current limitations on genome‐wide epigenetic profiling by incorporating fluorescence‐activated cell sorting and sample‐specific barcoding to examine cell‐type‐specific CpG methylation in discrete brain regions of individuals. We demonstrate the value of this method by characterizing differences in 5‐methylcytosine (5mC) in neurons and non‐neurons of the ventromedial prefrontal cortex of individual adult C57BL/6 mice, using as little as 50 ng of genomic DNA per sample. We find that the neuronal methylome is characterized by greater CpG methylation as well as the enrichment of 5mC within intergenic loci. In conclusion, MBD Ultra‐Seq is a robust method for detecting DNA methylation in neurons derived from discrete brain regions of individual animals. This protocol will facilitate the detection of experience‐dependent changes in DNA methylation in a variety of behavioural paradigms and help identify aberrant experience‐induced DNA methylation that may underlie risk and resiliency to neuropsychiatric disease.