Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions of cells. Through the development of a miniaturized genome-scale chromatin immunoprecipitation method (miniChIP–chip), we have documented the genome-wide chromatin states of low abundant populations that comprise hematopoietic stem cells and immediate progeny residing in murine bone marrow. In this report, we describe the miniChIP methodology that can be used for increasing an understanding of the epigenetic mechanisms underlying hematopoietic stem and progenitor cell function. Application of this method will reveal the contribution of dynamic chromatin structure in regulating the function of other somatic stem cell populations, and how this process becomes perturbed in pathological conditions.
Miniaturized chromatin immunoprecipitation assays; Microarray technology; Histone modifications; Stem and progenitor cells; Epigenetic regulation; Lineage commitment
Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets.
Our results indicate that accurate measurement of siRNA efficacy by RT-qPCR requires careful attention for the selection of the primers used to amplify the target EGFR mRNA.
We conclude that when assessing siRNA efficacy with RT-qPCR, more than one primer set targeting different regions of the mRNA should be evaluated and at least one of these primer sets should amplify a region encompassing the siRNA recognition sequence.
NF-κB regulates several important expressions, such as cytokine release, anti-apoptosis, adhesion molecule expression, and cell cycle processing. Several NF-κB inhibitors have been discovered as an anti-tumor or anti-inflammatory drug. The activity of NF-κB transcription factor is negatively regulated by IκB binding. In this study, IκB assay system was established and IκB–EGFP fusion protein was used as an indicator to monitor the effects of substances on the IκB degradation. The results indicated that the chosen hydroquinone could inhibit the IκB degradation and cause the cell de-attachment from the bottom of culture plate. In addition, this system could also monitor the IκB degradation of microbial metabolite of natural mixtures of propolis. Thus, the IκB assay system may be a good system for drug discovery related to microbial metabolite.
Microbial metabolite; Antioxidant; IκB; EGFP; Hydroquinone; Propolis
The use of architectural and morphological characteristics of cells for establishing prognostic indicators by which individual pathologies are assigned grade and stage is a well-accepted practice. Advances in automated micro- and macroscopic image acquisition and digital image analysis have created new opportunities in the field of prognostic assessment; but, one area in experimental pathology, animal models for colon cancer, has not taken advantage of these opportunities. This situation is primarily due to the methods available to evaluate the colon of the rodent for the presence of premalignant and malignant pathologies. We report a new method for the excision and processing of the entire colon of the rat and illustrate how this procedure permitted the quantitative assessment of aberrant crypt foci (ACF), a premalignant colon pathology, for characteristics consistent with progression to malignancy. ACF were detected by methylene blue staining and subjected to quantitative morphometric analysis. Colons were then restained with high iron diamine–alcian blue for assessment of mucin depletion using an image overlay to associate morphometric data with mucin depletion. The subsequent evaluation of ACF for beta-catenin staining is also demonstrated. The methods described are particularly relevant to the screening of compounds for cancer chemopreventive activity.
colon carcinogenesis; aberrant crypt foci; mucin depletion; beta-catenin; image analysis; morphometry
There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.
transcription factor; microsphere-based immunoassay; NF-κB; HIF-1
Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide) (EBI) to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.
colchicine-binding site inhibitor; taxol-binding site inhibitor; N,N'-ethylene-bis(iodoacetamide); EBI; tubulin affinity assay; antimicrotubule agent
Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.
Lentiviral vector; Indoleamine 2; 3 dioxygenase; Primary fibroblast; Transplantation; Immunogenicity
Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protocol to purify, fractionate, blot, and hybridize Streptomyces RNA. It is based on grinding by a high-performance laboratory ball mill, followed by prompt lysis with acid phenol-guanidinium, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.
streptomycetes; total RNA purification; RNA processing; RNA degradation; RNA glyoxylation; alkaline blotting; northern hybridization
Laser capture microdissection (LCM) enables collection of cell populations highly enriched for specific cell types that have the potential of yielding critical information about physiological and pathophysiological processes. One use of cells collected by LCM is for gene expression profiling. Samples intended for transcript analyses should be of the highest quality possible. RNA degradation is an ever-present concern in molecular biological assays, and LCM is no exception. This paper identifies issues related to preparation, collection, and processing in a lipid-rich tissue, rodent mammary gland, in which the epithelial to stromal cell ratio is low and the stromal component is primarily adipocytes, a situation that presents numerous technical challenges for high-quality RNA isolation. Our goal was to improve the procedure so that a greater probe set present call rate would be obtained when isolated RNA was evaluated using Affymetrix microarrays. The results showed that the quality of RNA isolated from epithelial cells of both mammary gland and mammary adenocarcinomas was high with a probe set present call rate of 65% and a high signal-to-noise ratio.
Laser capture microdissection; Mammary epithelial cells; RNA quality
The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.
Blood Cells; Bone Marrow Cells; Cell Proliferation; Cell Survival; Mesenchymal Stem Cells; Physiologic Neovascularization