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At least 289 records · Page 16Linked to original sources
PubMed2002

Gene expression profiles in the brain from phencyclidine-treated mouse by using DNA microarray.

Recently DNA microarray technology has been introduced into analyses of comprehensive biological functions. This DNA microarray is a new technology for simultaneous analysis to examine expression patterns of thousands of genes. It was thought that this technique should be very useful for examination of cellular and molecular mechanisms of drugs of abuse: cocaine, amphetamine, and others. This technology was therefore applied for the rapid analysis of gene expression in the brain from phencyclidine-treated mice. Mainly mouse DNA microarray was examined by using labeled cDNAs produced from a control mouse brain mRNA and from brain mRNA of mouse exposed to drugs as probes. Some changes in a probe from brain mRNA of drug-treated mouse could be observed, but it was necessary to examine another DNA microarray, including more samples from the brain.

Animals↗
PubMed2002

DNA vaccination of ferrets with chimeric influenza A virus hemagglutinin (H3) genes.

Recently a technology was established based on homologous recombination that allowed the rapid generation of chimeric HA genes of influenza viruses, containing the antigenic determinants obtained from various influenza virus A (H3N2) viruses. In the present report plasmids were generated using a H3 HA vector handle and the hypervariable regions of two genetically distinct influenza A H3N2 viruses, A/Stockholm/7/97 and A/Netherlands/18/94. In a ferret model it was shown that immunisation with plasmid DNA encoding chimeric HA indeed elicited antibody responses specific for the virus from which the hypervariable region with the antigenic determinants were obtained. After DNA-immunisation of the ferrets, protective immunity against infection with influenza virus A/Netherlands/18/94 was evaluated.

Animals↗
PubMed1997

DNA vaccines: safety and efficacy issues.

DNA technology has been harnessed to produce a variety of plasmid-based vaccines designed to prevent viral, bacterial and parasitic infections. The rapid adoption and implementation of this novel vaccine strategy carries with it important safety and efficacy concerns. This review will focus on whether DNA vaccines (1) are likely to induce systemic or organ-specific autoimmune disease, (2) have the potential to induce tolerance rather than immunity, and (3) are as effective in individuals with depressed immune function as they are in healthy adults.

Aging↗
PubMed1992

Genome research: fulfilling the public's expectations for knowledge and commercialization.

This article provides a historical perspective for the patenting of gene sequences and describes the fundamentals and evolution of patent law. It summarizes federal technology transfer law and policy and assesses the impacts of patenting on academic research. The patentability of gene sequences is then considered along with potential impacts that published sequence data may have on obtaining patent protection for downstream products. Industry's position on gene patenting is summarized and perspectives from the emerging public record on these issues are presented. The article discussing points at which the filing of patent applications and the licensing of patents may be appropriate. It concludes that technology transfer policies for genome research must be adopted carefully so that they remain viable in a time of rapid technological change.

Animals↗
PubMed2025

CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells.

The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.

Humans↗
PubMed2001

Purification of eukaryotic MutL homologs from Saccharomyces cerevisiae using self-affinity technology.

Self-cleaving affinity technology is an effective tool for rapid purification of native sequence recombinant proteins overproduced in Escherichia coli. In this report, we describe the adaptation of this technology to purify DNA mismatch repair proteins overproduced in the eukaryote Saccharomyces cerevisiae. Mlh1 and Pms1 are homologs of the E. coli MutL protein that participate in a variety of DNA transactions in cells, including correction of DNA replication errors, recombination, excision repair, and checkpoint control. Difficulties in preparing substantial quantities of highly purified MutL homologs have impeded descriptions of their biophysical and biochemical properties and mechanisms of action. To overcome this limitation, here we use self-cleaving affinity technology to purify to apparent homogeneity the yeast Mlh1--Pms1 heterodimer and the individual yeast and human Mlh1 subunit. The availability of these proteins should accelerate an understanding of their multiple functions in mismatch repair and other DNA transactions. The general approach is a valid alternative for simple, rapid purification of recombinant proteins in yeast when expression in bacteria is unsuitable.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The diagnosis of tuberculosis: what's old, what's new.

The approach to the diagnosis of both active tuberculosis and latent infection has changed very little in the past several decades. For active disease, sputum smears with or without chest radiographs to aid in diagnostic accuracy, form the cornerstone of the diagnostic approach in many high-burden countries. These tests usually are supplemented by cultures when resources permit. The diagnosis of latent infection still relies on the use of the tuberculin skin test using purified protein derivative. The current global tuberculosis epidemic, which features large numbers of patients with human immunodeficiency virus infection and increasing rates of multidrug-resistant tuberculosis, makes accurate and rapid diagnosis of tuberculosis more urgent than ever before. Currently available technologies, most involving techniques of DNA amplification, can substantially improve the accuracy of the diagnosis of tuberculosis, although the use of such assays has been sharply limited because of concerns about cost. However, economic analyses suggest that these assays can be cost effective if they lead to sharp reductions in transmission through earlier treatment of infectious cases.

DNA, Bacterial↗
PubMed2004

Fetal blood group genotyping from DNA from maternal plasma: an important advance in the management and prevention of haemolytic disease of the fetus and newborn.

The cloning of blood group genes and subsequent identification of the molecular bases of blood group polymorphisms has made it possible to predict blood group phenotypes from DNA with a reasonable degree of accuracy. The major application of this technology, which has now become the standard of care, is the determination of a fetal RHD genotype in women with anti-D, to assess whether the fetus is at risk of haemolytic disease of the fetus and newborn (HDFN). Initially, the procurement of fetal DNA required the invasive procedures of amniocentesis or chorionic villus sampling. Since the discovery of fetal DNA in maternal plasma in 1997, the technology of detecting an RHD gene in this very small quantity of fetal DNA has developed rapidly, so that non-invasive fetal D typing can now be provided as a diagnostic service for D-negative pregnant women with anti-D. Within a few years, it is probable that fetuses of all D-negative pregnant women will be tested for RHD, to establish whether the mother requires antenatal anti-D immunoglobulin prophylaxis.

Blood Group Antigens↗
PubMed2002

Reverse vaccinology: a genome-based approach for vaccine development.

During the last century several approaches have been followed for the development of vaccines. These include live-attenuated viruses and bacteria, killed microorganisms and the subunit vaccines [1]. With the introduction of recombinant DNA technologies, new approaches have been exploited for vaccine manufacturing. However, the major problem remains the rapid identification of highly immunogenic and protective antigens suitable for vaccine development, which still relies on standard biochemical and microbiological techniques. The advent of genomics has greatly contributed to providing a new impulse to the microbial field. The complete genomic sequence of a human pathogen represents a new unexploited field, to be used for the design of novel vaccines and antimicrobial drugs. In the case of meningococcus B, four decades of continuous efforts, using conventional technologies of purifying antigens from the microorganism, had not been sufficient to deliver an effective and universal vaccine. It was therefore decided to obtain the genomic sequence of serogroup B Neisseria meningitidis (MenB) and use this information to identify vaccine candidates. This approach was named "reverse vaccinology"[2].

Animals↗
PubMedDate not supplied

Plasma desorption mass spectrometry of natural and recombinant peptides and proteins.

Plasma desorption mass spectrometry (PDMS), which utilizes the fission fragments from the spontaneous decay of californium-252 to ionize large biomolecules, has emerged as a powerful analytical tool in the field of protein chemistry. Because of its high mass range and sensitivity, PDMS is an excellent method for direct molecular weight determination of peptides and small proteins, with much better accuracy than that of the most commonly used classical methods of gel permeation chromatography or SDS-gel electrophoresis. Furthermore, PDMS analysis of the peptide mixture resulting from a specific enzymatic or chemical cleavage of the protein, allows rapid and facile checking of DNA-derived protein sequences and protein structures produced by recombinant DNA technology. The relatively nondestructive nature of the PD mass spectrometric analysis allows further confirmation of the sequence assignments of individual peptide fragments through additional chemical or enzymatic reactions on the PDMS matrix-bound peptides. This PD mapping approach, combined with classical gas phase sequencing, can be used to identify and locate post-translational modifications in proteins, such as glycosylation, phosphorylation, disulfide linkages, and also detect the presence of peptide and protein variants in synthetic, native and recombinant peptides and proteins.

Amino Acid Sequence↗
PubMed2001

Tissue microarray technology for high-throughput molecular profiling of cancer.

Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. The technique facilitates rapid translation of molecular discoveries to clinical applications. By revealing the cellular localization, prevalence and clinical significance of candidate genes, TMAs are ideally suitable for genomics-based diagnostic and drug target discovery. TMAs have a number of advantages compared with conventional techniques. The speed of molecular analyses is increased by more than 100-fold, precious tissues are not destroyed and a very large number of molecular targets can be analyzed from consecutive TMA sections. The ability to study archival tissue specimens is an important advantage as such specimens are usually not applicable in other high-throughput genomic and proteomic surveys. Construction and analysis of TMAs can be automated, increasing the throughput even further. Most of the applications of the TMA technology have come from the field of cancer research. Examples include analysis of the frequency of molecular alterations in large tumor materials, exploration of tumor progression, identification of predictive or prognostic factors and validation of newly discovered genes as diagnostic and therapeutic targets.

Animals↗
PubMed2004

Production of bioproducts through the use of transgenic animal models.

Transgenic livestock that produce recombinant proteins in their milk can provide an economic and safe system for production of valuable proteins, such as pharmaceutical proteins for treatment or prevention of human disease or biomaterials for medical use. This method of production is frequently referred to as biopharming. The promise of biopharming, that is the actual commercial production of pharmaceuticals and other bioproducts, is nearing fulfillment. Improvements in molecular and reproductive techniques and strong economic incentives have continued to drive the implementation of transgenic technology to domestic animals. Nuclear transfer using transgenic donor cells is rapidly becoming the predominant technique used in the production of transgenic livestock, replacing the direct injection of DNA into the zygotic pronuclei. Production of transgenic founder animals by nuclear transfer in combination with traditional reproductive technologies can result in the propagation of transgenic herds of sufficient size to meet market demands for commercially important proteins. While some of the companies that have established transgenic programs have run into setbacks owing to a combination of economic, scientific and regulatory difficulties, other companies are continuing to make significant advances. While further improvements are needed to increase efficiencies of production, economically viable production of recombinant proteins using livestock species is not only possible but should be a commercial reality in the very near future.

Animals↗
PubMed2001

Microfabricated capillary array electrophoresis DNA analysis systems.

Microfabricated "laboratory-on-a-chip" systems are revolutionizing all aspects of genetic analysis. The development of capillary array electrophoresis (CAE) microchannel plate devices makes possible the performance of 96 or more high-speed separations in parallel on a single wafer-scale device. The fluorescently labeled DNA samples are detected within the microchannels with a novel four-color rotary confocal fluorescence scanner. The capabilities of this system for genotyping are demonstrated through multiplex separations of short tandem repeat and hereditary haemochromatosis allele-specific amplicons. Furthermore, with newly developed folded channel designs that maintain high resolution, these CAE microplate systems are used to perform 96 high-quality DNA sequencing separations in parallel to approximately 500 bases per capillary in less than 30 min. These densely packed microfabricated device technologies will facilitate the even more rapid collection of vast amounts of genetic data in the future.

Base Sequence↗
PubMed2004

Molecular biology and DNA microarray technology for microbial quality monitoring of water.

Public concern over polluted water is a major environmental issue worldwide. Microbial contamination of water arguably represents the most significant risk to human health on a global scale. An important challenge in modern water microbial quality monitoring is the rapid, specific, and sensitive detection of microbial indicators and waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. Rapid microbiological analyses and detection of rare events in water systems are important challenges in water safety assessment since culture methods present serious limitations from both quantitative and qualitative points of view. To circumvent lengthy culture methods, newer enzymatic, immunological, and genetic methods are being developed as an alternative. DNA microarray technology is a new and promising tool that allows the detection of several hundred or even thousands DNA sequences simultaneously. Recent advances in sample processing and DNA microarray technologies provide new perspectives to assess microbial water quality. The aims of this review are to (1) summarize what is currently known about microbial indicators, (2) describe the most important waterborne pathogens, (3) present molecular methods used to monitor the presence of pathogens in water, and (4) show the potential of DNA microarrays in water quality monitoring.

Animals↗
PubMed1990

Mechanisms and consequences of mutation induction in mammalian cells.

Mutations have been studied for several decades in order to understand biological processes of great significance and the selection of better-adapted species. Our knowledge both of mutation spectra induced by genotoxic agents and the mechanisms involved in DNA damage processing is more advanced in bacteria than in animal cells. However, the use of new technologies such as shuttle vectors or the polymerase chain reaction will undoubtedly allow rapid progress in the next few years. Shuttle vectors consist of target sequences for monitoring mutagenic activity and additional sequences permitting DNA replication and selection, both in bacteria and in mammalian cells. These plasmids are very efficient in allowing the production of mutation spectra of a particular genotoxin in animal cells. In most cases, base substitutions occur predominantly at the sites of base damage and the type of substitution depends on the kind of damage. This has been well characterized using ultraviolet (UV) light as a mutagen. UV-induced mutations are targeted opposite pyrimidine-pyrimidine sites, where the two major UV lesions are produced. The direct relationships existing between mutation and cancer are exemplified by some hereditary diseases where deficiency in an enzymatic repair system is linked to a high incidence of tumours. Similarly, activation of some cellular proto-oncogenes occurs via specific point mutations. A correlation does exist between the mutation spectra found in model systems and the specific mutation found in the activated oncogene in tumours induced by a given genotoxin. This is particularly well illustrated in the DNA repair deficiency syndrome, xeroderma pigmentosum. The specific mutations found in activated ras oncogenes isolated from UV-stimulated skin tumours correlate well with the mutagenic properties of unrepaired UV-induced DNA lesions.

Animals↗
PubMed1993

The rapid analysis of dystrophin gene deletions shows variable electrophoretic mobility.

The introduction of PCR technology to the molecular diagnosis of genetic diseases has increased the speed and range of DNA tests available. Previous analyses of dystrophin gene mutations were time consuming, taking weeks to complete, and used radioisotopic methods. Further developments in DNA amplification and post-amplification techniques have made conventional tube PCR redundant. The rapid methodologies described enable the efficient screening of large populations for genetic disorders, although precautions must be taken when analysing the PCR products.

Cross-Linking Reagents↗
PubMed1999

Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation.

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.

Animals↗