High Stringency Washes Research Articles

A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

Double In situ Hybridization for MicroRNAs and mRNAs in Brain Tissues

MicroRNAs (miRNAs) participate in a variety of functions in the brain. Understanding the in vivo localization of miRNAs is an important step for uncovering their roles in brain function. However, the in situ detection of low-abundance miRNAs in brain tissues remains difficult and requires extensive optimization of in situ hybridization (ISH) protocols in individual laboratories. Thus, detailed information regarding experimental conditions would serve as a useful reference for researchers in this field. Here, we investigated and summarized the effects of adjusting a series of critical steps, including tissue fixation, probe accessibility and hybridization stringency, to standardize the currently used miRNA ISH procedures. As a result, we successfully detected several low-abundance miRNAs by ISH using the following experimental conditions: (1) use of fresh brain tissues, (2) digestion of brain samples with proteinase K, (3) LNA-probe hybridization at a temperature 37°C below the melting temperature of the RNA, (4) performance of high-stringency wash steps using 50% formamide in 1 × standard saline citrate (SSC) buffer. RT-PCR of the punched-out tissues using TaqManTM primers confirmed the ISH results. Finally, double-fluorescence ISH successfully demonstrated the colocalization of miRNAs and mRNAs. Thus, the detailed information regarding the miRNA ISH procedures used in this study may help to resolve the technical hurdles observed in the in vivo localization of miRNAs, and the elucidation of the specific roles of miRNAs.

Development of positive control tissue for in situ hybridisation using Alvetex scaffolds.

In situ hybridisation (ISH) is a robust method to determine the presence of mRNA for specific genes within a tissue. Ideally, positive and negative control tissues are used to determine probe specificity. However, this is not always possible, particularly for human genes where no knock-out controls exist. Here we report a novel method of growing positive control cells in a scaffold (Alvetex) to create 3D tissues suitable for sectioning with a cryostat. Sectioning slices through cells, similar to the effect on tissue and therefore provides improved penetration of the in situ riboprobes. ISH conducted on cells has been problematic due to the difficulty of efficient probe penetration, due to a semi-intact cell membrane, and cell preparations failing to withstand high stringency washes. Our new method circumvents this issue by enabling the positive control cells to be sectioned like a tissue. HEK cells transfected with the genes of interest (in this case CB1 and NeuN) grown in Alvetex and cryosectioned were utilised to validate riboprobes and establish stringency conditions. These conditions were then transferred directly to human brain sections. This method can be adapted to generate positive controls for ISH for any gene of interest. It provides a valuable option in human neuroscience where access to precious brain tissue is limited or where expression of a target gene is unknown.

Abstract P3-05-06: Automated analysis of Her2 FISH using combined Immunofluorescence and FISH signals

Abstract Introduction: Qualifying patients for Her2 targeted therapy is currently done by detecting Her2 protein overexpression or gene amplification using immunohistochemistry and/or FISH. We have recently developed a method for detecting both signals on the same tissue section allowing direct correlation of protein expression and gene copy number on a cell by cell basis. Accurate assessment of Her2 gene copy number is critical and can pose a challenge due to tumor heterogeneity. This paper reports the accuracy of a proprietary FISH dot counting algorithm on a cell-by-cell basis, potentially allowing analysis of thousands instead of dozens of tumor cells. Method: Automatic FISH signal counts were compared to manual counts of 888 cells selected from 19 invasive ductal breast carcinoma samples exhibiting varying degrees of Her2 expression collected between June 2011 and March 2012. Tissue sections (4 µm) were mounted on positively charged slides, baked and processed through deparaffinization, rehydration and antigen retrieval, then stained for immunofluorescence (IF) using Cy5 labeled Her2 and Cy3 labeled cytokeratin antibodies, counterstained with DAPI, and imaged using InCell 2000 analyzer with GE-proprietary acquisition and processing software. Images were collected at 10x magnification and digitally stitched to span the entire tissue section. A pathologist then selected separate tumor and adjacent normal epithelium regions for subsequent imaging at 40x magnification. Slides were subsequently processed for FISH by pepsin digestion and then subjected to FISH by using PathVysion kit (Abbott Molecular, Des Plaines, IL). After hybridization and subsequent high stringency washes, samples were DAPI stained and mounted for microscopy. Samples were imaged at 40x at the same regions recorded for 40x IF acquisition, using filtersets appropriate for FISH fluorophores and DAPI. A proprietary automated processing algorithm was used to analyze combined IF and FISH signals and derive case specific Her2 score from the tumor and/or adjacent normal epithelium. Cell-level dot counting accuracy was assessed using two metrics comparing automated counts to manual counts: cell classification agreement, where a normal cell was defined as having 3 or less Her2 and Cep17 dots; and dot-counting match, where a difference of more than 20% in absolute counts was considered an error. Result: Our automatic results gave an overall cell-by-cell classification agreement of 88% (range 71% to 98% by case). Combining classification agreement and counting match, our algorithm gave an overall accuracy of 81% (range 63% to 97% by case). Restricting to tumor tissues (as judged by pathologist review of IF) classification agreement and accuracy were 84% and 72%, respectively. Conclusion: The observed variability in algorithm performance between the different cases was due to the fact that error root causes were case dependent. For instance, the main cause of over-counting errors was image noise and artifacts. On the other hand, the main cause of under-counting was low image contrast, especially in highly amplified cases. These results are an early indication of the promise of automatic dot counting applied to breast cancer slides multiplexed for Her2 IF and FISH. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-06.

Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach

Many protein interaction domains bind short peptides based on canonical sequence consensus motifs. Here we report the development of a peptide array-based proteomics tool to identify proteins directly interacting with ligand peptides from cell lysates. Array-formatted bait peptides containing an amino acid-derived cross-linker are photo-induced to crosslink with interacting proteins from lysates of interest. Indirect associations are removed by high stringency washes under denaturing conditions. Covalently trapped proteins are subsequently identified by LC-MS/MS and screened by cluster analysis and domain scanning. We apply this methodology to peptides with different proline-containing consensus sequences and show successful identifications from brain lysates of known and novel proteins containing polyproline motif-binding domains such as EH, EVH1, SH3, WW domains. These results suggest the capacity of arrayed peptide ligands to capture and subsequently identify proteins by mass spectrometry is relatively broad and robust. Additionally, the approach is rapid and applicable to cell or tissue fractions from any source, making the approach a flexible tool for initial protein-protein interaction discovery.

Electrochemical biosensors employing an internal electrode attachment site and achieving reversible, high gain detection of specific nucleic acid sequences.

Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives), or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ∼800% at saturating target, a detection limit of just 50 pM, and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus, these devices are fully reusable.

Perspectives on Utilizing Unique Features of Microfluidics Technology for Particle and Cell Sorting

Sample preparation is often the most tedious and demanding step in an assay, but it also plays an essential role in determining the quality of results. As biological questions and analytical methods become increasingly sophisticated, there is a rapidly growing need for systems that can reliably and reproducibly separate cells and particles with high purity, throughput and recovery. Microfluidics technology represents a compelling approach in this regard, allowing precise control of separation forces for high performance separation in inexpensive, or even disposable, devices. In addition, microfluidics technology enables the fabrication of arrayed and integrated systems that operate either in parallel or in tandem, in a capacity that would be difficult to achieve in macro-scale systems. In this report, we use recent examples from our work to illustrate the potential of microfluidic cell- and particle-sorting devices. We demonstrate the potential of chip-based high-gradient magnetophoresis that enable high-purity separation through reversible trapping of target particles paired with high-stringency washing with minimal loss. We also describe our work in the development of devices that perform simultaneous multi-target sorting, either through precise control of magnetic and fluidic forces or through the integration of multiple actuation forces into a single monolithic device. We believe that such devices may serve as a powerful "front-end" module of highly integrated analytical platforms capable of providing actionable diagnostic information directly from crude, unprocessed samples - the success of such systems may hold the key to advancing point-of-care diagnostics and personalized medicine.

Comparison of mealybug (Planococcus lilacinus) and fruit fly genomes: isolation and analysis of conserved sequences and their utility in studying synteny in the mealybug

By using ligation-mediated PCR products from mealybug DNA as tester and biotinylated fly DNA as driver, we recovered a fraction of the tester that remains hybridized to driver following high-stringency washing conditions. This fraction is expected to contain mealybug sequences conserved in the fly (MCF). Reciprocal experiments enabled the isolation of fly sequences conserved in the mealybug (FCM). Coding sequences among MCF show amino acid identities >40% with fly proteins, allowing a reliable identification of orthologs. Three sequences from the fly cytogenetic positions 98–99 were hybridized onto mealybug chromosomes and the results identified differences in synteny between the two species. Taken together, our results present a method for direct isolation of sequences conserved between an ‘orphan’ (mealybug) genome and a ‘reference’ (fly) genome and showed that these sequences can be used to study chromosome synteny in the mealybug.

Protease Nexin-1, tPA, and PAI-1 are Upregulated in Cryoglobulinemic Membranoproliferative Glomerulonephritis

Thymic stromal lymphopoietin (TSLP) transgenic mice develop cryoglobulin-associated membranoproliferative glomerulonephritis, characterized by renal monocyte/macrophage infiltration, marked expansion of extracellular matrix, and variable intraluminal and mesangial deposits of cryoglobulins. A microarray approach was used to study global gene expression in glomerular RNA obtained from these mice, as well as from combined TSLP transgenic and Fcγ receptor IIb null mice (TSLP/FcIIb−/−), which develop aggravated membranoproliferative glomerulonephritis. Protease nexin-1 (PN-1) and tissue plasminogen activator (tPA), two potential regulators of fibrosis that are involved in the fibrinolytic and coagulation pathways, were dramatically upregulated in TSLP mice compared with wild-type controls. In situ hybridization revealed minimal expression of PN-1 mRNA in the glomeruli of wild-type mice, increased expression in TSLP mice, and the greatest expression in the mesangial cells of TSLP/FcIIb−/− mice. Immunohistochemistry demonstrated greater expression of PN-1, tPA, and PAI-1 in the mesangial cells of TSLP mice compared with wild-type and the greatest in TSLP/FcIIb−/− mice. In cultured mesangial cells, incubation with cryoglobulins induced an upregulation of PN-1 mRNA; increased expression of PN-1, tPA, and PAI-1 proteins; and stimulated secretion of TGF-β1. It is concluded that PN-1, tPA, PAI-1, and TGF-β1 are likely important mediators of murine cryoglobulinemic glomerulonephritis and that the cryoglobulins may directly upregulate their expression.

Dermokine: An Extensively Differentially Spliced Gene Expressed in Epithelial Cells

Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.

An Antisense Transcript Induced by Wnt/β-Catenin Signaling Decreases E2F4

Wnt signaling induces the nuclear accumulation of beta-catenin and transcription of specific target genes via the DNA-binding proteins TCF/Lef. Although all known beta-catenin target genes encode proteins, genome-wide RNA profiling studies indicate that many transcripts do not have this capability. Transcription factor-binding sites associated with these noncoding transcripts can be identified using unbiased techniques such as serial analysis of chromatin occupancy (SACO). We used this method to identify a beta-catenin-regulated antisense RNA expressed in HCT116 colorectal carcinoma cells, a cellular model of activated beta-catenin signaling. Genomic signature tags designating putative beta-catenin-binding sites mapped to the 3'-untranslated region (3'-UTR) of the E2F4 gene. We showed that both beta-catenin and TCF4 bind to the E2F4 3'-UTR site in vivo, inducing expression of an E2F4 antisense transcript. LiCl, which mimics Wnt signaling, also induced expression of the E2F4 antisense transcript and decreased E2F4 protein levels. This effect was blocked by a cDNA expressing the E2F4 3'-UTR sense strand. The antisense-mediated decrease in E2F4 protein was reflected by reduced E2F4 association with specific target genes, including CCNA2, CDC2, PCNA, and Rad54. We propose that Wnt/beta-catenin signaling may contribute to colorectal carcinogenesis by reducing the level of the E2F4 cell cycle repressor via an antisense mechanism.

CKAP4/p63 Is a Receptor for the Frizzled-8 Protein-related Antiproliferative Factor from Interstitial Cystitis Patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

Dephosphorylation and Genome-Wide Association of Maf1 with Pol III-Transcribed Genes during Repression

Nutrient deprivation and various stress conditions repress RNA polymerase III (Pol III) transcription in S. cerevisiae. The signaling pathways that relay stress and nutrient conditions converge on the conserved protein Maf1, but how Maf1 integrates environmental conditions and couples them to transcriptional repression is largely unknown. Here, we demonstrate that Maf1 is phosphorylated in favorable conditions, whereas diverse unfavorable conditions lead to rapid Maf1 dephosphorylation, nuclear localization, physical association of dephosphorylated Maf1 with Pol III, and Maf1 targeting to Pol III-transcribed genes genome wide. Furthermore, Maf1 mutants defective in full dephosphorylation display maf1Delta phenotypes and are compromised for both nuclear localization and Pol III association. Repression conditions also promote TFIIIB-TFIIIC interactions in crosslinked chromatin. Taken together, Maf1 appears to integrate environmental conditions and signaling pathways through its phosphorylation state, with stress leading to dephosphorylation, association with Pol III at target loci, alterations in basal factor interactions, and transcriptional repression.

Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.

Oxidative Stress-mediated, Post-translational Loss of MafA Protein as a Contributing Mechanism to Loss of Insulin Gene Expression in Glucotoxic Beta Cells

Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. Recently, RIPE-3b1 activator was cloned and identified as the mammalian homologue of avian MafA/Maf-L (MafA). This enabled us to carry out more extensive studies of the role of MafA in glucotoxicity than were hitherto possible. Northern analysis of glucotoxic HIT-T15 cells revealed normal amounts of MafA mRNA, but Western analysis demonstrated a 97 +/- 1% reduction in MafA protein (p < 0.0001). The proteasome is a likely site for MafA degradation as lactacystin, an irreversible proteasome inhibitor, caused an accumulation of MafA protein. Antioxidants have previously been shown to prevent the adverse effects of glucose toxicity on beta cell function both in vivo and in vitro. In the current study, chronic culturing of HIT-T15 cells with the antioxidant N-acetylcysteine (NAC) prevented loss of MafA protein (late passage = 18.9 +/- 10.4% of early passage, p < 0.001; late passage with NAC = 68.7 +/- 19.7% of early passage, p = not significant) and loss of DNA binding (late passage = 63.7 +/- 9% of early passage, p < 0.02; late passage with NAC = 116 +/- 10% of early passage, p = not significant). Additionally, transient transfection of PDX-1 or MafA cDNA into glucotoxic cells increased PDX-1 and MafA protein levels and individually increased insulin promoter activity (untreated = 34%, PDX-1 = 70%, MafA = 78%; percentage of activity of early passage cells), whereas the combined transfection of MafA and PDX-1 completely restored insulin promoter activity. This recovery of promoter activity following transient transfection had no effect on endogenous insulin mRNA. However, adenoviral infection of MafA and PDX-1 significantly increased endogenous insulin mRNA levels by 93% (121 +/- 9 versus 233 +/- 18 density light units; n = 5, p < 0.001). We conclude that the absence of MafA protein from beta cells via chronic oxidative stress contributes importantly to the loss of endogenous insulin gene expression as glucose toxicity develops.

Application of microchip assay system for the measurement of C-reactive protein in human saliva

In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in this study to provide direct experimental evidence that chronic periodontal disease may be associated with higher levels of salivary CRP.

Targeted Gene Delivery to Human Airway Epithelial Cells with Synthetic Vectors Incorporating Novel Targeting Peptides Selected by Phage Display

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including R/KSM, LP/QHK and PSGA/TARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.

Importance of TRF1 for Functional Telomere Structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.

Quantitative in situ hybridization for peptide mRNAs in mouse brain

The objective was to determine the feasibility of using a radioactive capture method (Fuji FLA 2000) and image analysis system for the measurement of peptide mRNA levels in specific brain regions in mice. As a test mRNA, we chose vasopressin (VP) and oxytocin (OT) because they are expressed in abundance in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). A comparison was made between free-floating and slide-mounted sections to determine which method yielded better results. Mouse brains were fixed in 4% paraformaldehyde (PFA) and processed for in situ hybridization using 35S-oligonucleotide probes for VP and OT. After overnight hybridization and high stringency washes, 25-μm brain sections and 14C standards were exposed to a BAS-IIIs Fuji imaging film over a range of times (4 h–6 days). Results showed that there was an intense hybridization reaction in the PVN and SON, making it possible to distinguish the specific brain regions. Using Image Gauge Software, the signal was quantified in PVN and SON. A comparison of the different exposure times showed that the signal could be measured after as little as 4 h. The intensity readings increased over time while the calculated radioactivity remained constant. The free-floating method was superior to the slide-based system, providing a lower background and a higher signal. The data illustrates the applicability of the phosphor imaging system for the reproducible measurement of mRNA levels in discrete regions of the mouse brain.

Gene expression changes associated with cytotoxicity identified using cDNA arrays.

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes.