PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Genome engineering”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 829 records · Page 46Linked to original sources
PubMed2003

Specific targeted integration of kanamycin resistance-associated nonselectable DNA in the genome of the yeast Saccharomyces cerevisiae.

Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration. To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA. These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated. The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments. With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag. This plasmid system provides for a new molecular tool to integrate any DNA fragment at any genome location in [cir+] yeast strains. Moreover, the system can be extrapolated to other eukaryotic cells in which the FLP/FRT system functions efficiently.

Cloning, Molecular↗
PubMed1994

The structure and function of the first component of complement: genetic engineering approach (a review).

The availability of cDNA and genomic clones for the subcomponents of C1, as well as the recognition of the modular organization of serine-proteases have opened up exciting new possibilities for approaching structural problems. In this review the latest achievements of combined protein engineering, functional and structural studies are summarized. The concept of this research is to construct deletion, point and hybrid mutants of the highly homologous C1r and C1s subcomponents, to reveal the functional role of individual modules, map the interaction sites between subcomponents of the C1 complex and refine the structural model of C1. The first prerequisite of such an approach was the expression of the subcomponents in a eukaryotic system, in biologically active form. This was followed by expression of various mutants. Autographa californica nuclear polyhedrosis virus was used as vector to express human C1r and C1s in Spodoptera frugiperda cell culture and in lepidopteran larvae. The yield of expression was high enough to isolate recombinant subcomponents for structural and functional studies. Recombinant viruses containing the A-, B-, and C-chains of C1q were also constructed. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. The results with deletion mutants of C1r show that both, domain I, and II are absolutely necessary for the tetramer formation and both have regulatory role in the autoactivation. The C1s alpha R hybrid does not dimerize in presence of Ca2+, however it can form a tetramer with C11(2) that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. The C1Rs hybrid behaves like C1r, providing haemolytically active C1 with C1q and C1s. This observations shows that the regulatory domains determine the high functional specificity of the serine-protease subcomponents of C1. In order to control the autoactivation process point mutant cDNAs were constructed by altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation.

Animals↗
PubMed2002

The art and design of genetic screens: Arabidopsis thaliana.

Molecular genetic studies rely on well-characterized organisms that can be easily manipulated. Arabidopsis thaliana--the model system of choice for plant biologists--allows efficient analysis of plant function, combining classical genetics with molecular biology. Although the complete sequence of the Arabidopsis genome allows the rapid discovery of the molecular basis of a characterized mutant, functional characterization of the Arabidopsis genome depends on well-designed forward genetic screens, which remain a powerful strategy to identify genes that are involved in many aspects of the plant life cycle.

Alleles↗
PubMed1993

Design of an object-oriented database for reverse genetics.

We present the design of an object-oriented database system for reverse genetics applications. Such a database will encapsulate not only the data in the genetic and physical maps, but also the methods used to create the maps as well as methods to link them to other databases, such as GenBank, PIR, and MedLine. The purpose of this database is to provide the fungal genetics community with an electronic tool for identifying the biochemical function of any DNA fragment in the database--electronic reverse genetics. Such a tool for reverse genetics will enable researchers to identify the biochemical functions associated with genes encoding proteins in fungal development pathways, purine metabolism, the heat shock response, and molecular chromosome mechanics and evolution. Our initial goal is to apply the database for the genome mapping of the filamentous fungi, Aspergillus nidulans and Neurospora crassa, at the University of Georgia and the University of Leeds in England.

Aspergillus nidulans↗
PubMed2006

Targeting the human genome.

In recent years, some useful nucleic-acid-based tools including antisense oligonucleotides, aptamers, ribozymes, and small interfering RNA have been developed to alter the expression of a given gene. To date, however, these methods have proven to be generally insufficient for many applications and typically have not demonstrated high delivery efficiency or high target specificity in vivo. Emerging technologies that employ artificially designed transcription factors could offer an alternative solution, as they can recognize target DNA sequences with high specificity. In addition, these artificial proteins can be used not only as transcriptional regulators but also as genome modifiers that cleave and stimulate mutations at desired positions in the genome. These nucleotide-targeting molecules must be delivered efficiently to the target cells to promote their therapeutic activity and several delivery technologies have been developed for this purpose.

Drug Delivery Systems↗
PubMed1996

Negative-strand RNA viruses: genetic engineering and applications.

The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.

Animals↗
PubMed2005

Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin.

To improve the production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633, two genetic engineering strategies were followed. Firstly, additional copies of subtilin self-protection (immunity) genes spaIFEG have been integrated into the genome of the producer strain. Their expression significantly enhanced the subtilin tolerance level, and concomitantly, the subtilin yield 1.7-fold. Secondly, a repressor of subtilin gene expression, the B. subtilis general transition state regulator protein AbrB, was deleted. A sixfold enhancement of the subtilin yield could be achieved with the abrB deletion mutant; however, the produced subtilin fraction predominantly consists of succinylated subtilin species with less antimicrobial activity compared to unmodified subtilin.

ATP-Binding Cassette Transporters↗
PubMed2006

Chloroplast genetic engineering via organogenesis or somatic embryogenesis.

Chloroplast genetic engineering offers a number of unique advantages, including high-level transgene expression, multigene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to other important plant species. For example, Arabidopsis may be an ideal model system for chloroplast functional genomics. The employment of chloroplast transformation technology in Arabidopsis has been hampered by the lack of an efficient and reproducible protocol that provides fertile chloroplast transgenic plants. Transformation of the Arabidopsis chloroplast genome was achieved via organogenesis but the efficiency was at least a 100-fold lower than in tobacco and had the drawback of polyploidy in the leaf tissue that resulted in sterile transgenic plants. This problem can be overcome by adapting procedures that are now available to regenerate plants from both diploid and tetraploid explants via callus. In addition, it is feasible to regenerate Arabidopsis via somatic embryogenesis. Recent breakthroughs in highly efficient plastid transformation of recalcitrant crops such as cotton and soybean have opened the possibility of engineering Arabidopsis plastid genome via somatic embryogenesis. Therefore, protocols of recent improvements in tissue culture, DNA delivery, and the novel vector designs are provided here in order to achieve highly efficient plastid transformation in Arabidopsis.

Arabidopsis↗
PubMed2002

A rapid and easy method for production and selection of recombinant adenovirus genomes.

Adenoviruses are used widely as vectors for gene therapy. Due to the large size of their genome there is a low frequency of unique restriction sites and many techniques have been described to construct recombinant viruses. Whatever the considered technique, the Escherichia coli strain BJ5183 is used to obtain recombinant adenovirus genomes in a plasmid, or to construct defective viral backbones which will be used to produce infectious viral particles by homologous recombination in HEK293 cells. Unfortunately BJ5183 bacteria do not produce a sufficient amount of plasmid DNA to allow for restriction analysis. Plasmids have to be transferred into another strain to detect the expected construction. It is reported now that the common E. coli strain, Top10F' can be used for the construction of recombinant adenovirus genomes. A plasmid carrying a kanamycin resistance gene and containing the two ends of the adenovirus genome was used. It permits modification by classical molecular biology techniques or homologous recombination at both ends of the genome. The remainder of the genome is introduced by homologous recombination in Top10F'. Several homologous recombination steps were successfully performed without the steps of extraction and introduction of plasmid DNA in another strain to check the plasmids obtained.

Adenoviruses, Human↗
PubMed2004

Genetic engineering in the mouse: tuning TNF/TNFR expression.

With the exponential increase in the number of genes identified by various genome projects, it has become imperative that efficient methods be developed for deciphering gene function. Genetically engineered strains of mice are now critical research tools for basic biomedical research and for genomic approaches for the development of new therapeutic treatments for human disease. Over the past ten years it has become possible to make essentially any mutation in the mouse by transgenesis and homologous recombination in embryonic stem cells. Current advances in the "genetic engineering of the mouse," including the tissue-specific activation or inactivation of gene expression combined with developing technologies for switching gene expression on and off at will, provide experimental settings unprecedented in their potential to offer answers to long-standing questions.

Alleles↗
PubMed1998

Increased infectivity of hepatitis A virus cDNA clones with engineered 5'-terminal extra-cistronic sequences.

The genomic RNA of Hepatitis A virus (HAV), a picornavirus of the hepatovirus group, is a single-stranded molecule, ca. 7.5 kb in length of positive polarity. Translation of this uncapped RNA starts at the 10th (or 11th) AUG triplet (position 734-36), by a mechanism of internal initiation of translation. The long sequences extending between the uncapped 5'-end and the translation initiation site contain two (instead of just one) pyrimidine-rich tracts (PRTs) spanning nucleotides 94-140 and 711-724, respectively. The latter lies only 11 nucleotides upstream from the initiation site of translation, and the question arose as to whether the notoriously poor replication ability of HAV was a consequence of a down regulation of translation due to the too short "spacer" sequence intervening between the 3'-PRT and the initiation of the main open reading frame. To address this issue, a series of full-length HAV cDNA clones were constructed in which the "spacer" sequence (normally 11 nts) was brought to 45 nts. Following transfection of COS-1 cells with these constructs, the amount of HAV (+)-strand RNA was determined by dot hybridization using a strand-specific RNA probe. HAV cDNA clones carrying a 45-nt "spacer" increased two-fold the rate of (+)-strand viral RNA synthesis, suggesting that the poor translation ability of HAV RNA may be one of the mechanisms responsible for the lengthy replication cycle of HAV.

Animals↗
PubMed2002

Plant biotechnology in agriculture.

Knowledge on plant genomes has progressed during the past few years. Two plant genomes, those of Arabidopsis thaliana and rice, have been sequenced. Our present knowledge of synteny also indicates that, despite plasticity contributing to the diversity of the plant genomes, the organization of genes is conserved within large sections of chromosomes. In parallel, novel plant transformation systems have been proposed, notably with regard to plastid transformation and the removal of selectable marker genes in transgenic plants. Furthermore, a number of recent works considerably widen the potential of plant biotechnology.

Agriculture↗
PubMed2004

Robust functional gene validation by adenoviral vectors: one-step Escherichia coli-derived recombinant adenoviral genome construction.

We describe here a clonal approach for efficient and robust construction of recombinant adenoviral genomes that holds certain advantages over existing approaches. Transgenes of interest are cloned into a small, conditionally replicating plasmid containing the left end of a recombinant adenoviral genome, encompassing pIX coding regions. Transformation of this plasmid into recombination-competent Escherichia coli bearing a plasmid containing the right end of a recombinant adenoviral genome, commencing from pIX coding regions, yields a stable co-integrated plasmid encoding a full adenoviral genome, by virtue of shared homology in pIX coding regions contained in both plasmids. The recombination process yielding the full adenoviral plasmid requires only one step, and always results in the formation of only the desired recombinant adenoviral genome. Thus, no screening is required to identify the correct plasmid encoding the desired recombinant adenoviral genome. In addition, the plasmid encoding the right-hand side of the adenoviral genome is itself incapable of producing contaminating adenovirus. We have successfully employed this approach to generate over 200 recombinant adenoviruses, obtaining only the desired recombinant adenoviral species each time. The process is amenable to medium-to-high-throughput parallel construction of adenoviral genomes, and as such should aid efforts aimed towards high-throughput functional annotation of therapeutic gene targets, which aim to leverage the benefits of adenoviruses as gene delivery and expression vectors.

Adenoviridae↗
PubMed2001

New genetics, new ethics? Globalisation and its discontents.

The paper discusses the rapid and significant development of new genetic technologies (in health, food and agriculture) in the theoretical context of the globalisation debate. We show how the studies on the ethical, legal and social implications of biotechnological innovation have themselves emerged as an important factor in the technological innovation and product development process. Ethical considerations are becoming integral to attempts to understand techno-scientific developments in late modernity. However, ethical studies have so far been more focused on the medical rather than the overall commercial application of genomics. Their relevance to this wider context of the globalised commodification of new genetics needs to be explored. Of special significance is the introduction of ethical considerations into the debates on globalised risk, which we will explore in relation to increasing disparities between the global North and South.

Biotechnology↗
PubMed2005

Construction and analysis of genomic, full-length infectious foamy virus DNA clones.

The molecular engineering of recombinant plasmid DNA clones containing the full-length and replication-competent feline foamy (retro)virus (FFV) proviral genome is described. The methods used to combine subgenomic FFV DNA fragments can be applied to other retrovirus genomes, resulting in full-length, bacterially cloned retroviruses. In addition, techniques used to determine the replication competence of the cloned viral genomes are described. Alternative technologies (not described here) are also discussed, together with the application of cloned genomes in modern molecular virology; for example, in functional genetic studies or as starting material for retrovirus vector development.

Animals↗
PubMed1990

Gene replacement and expression of foreign DNA in mycobacteria.

A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms.

Blotting, Southern↗