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Results for “Genome engineering”

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At least 217 records · Page 12Linked to original sources
PubMed1990

Genetic engineering of the pseudorabies virus genome to construct live vaccines.

Pseudorabies virus (PRV) is a herpesvirus of pigs. Homologous recombination with plasmids offers a method to engineer precise changes in the PRV genome to produce advantageous live vaccines. Safety can be ensured by using a non-reverting deletion to inactivate the thymidine kinase gene. One particularly important feature of new PRV vaccines is deletion of an antigen, so that vaccinated pigs are serologically distinguishable from infected pigs. We have constructed a live vaccine strain with deletions in the thymidine kinase gene and in the gene for a glycoprotein, gX. Molecular engineering techniques made it possible to choose deletion of gX, which has no known immunological significance, over deletion of other glycoproteins that contribute to protective immunity. Extensive experiments in pigs with isogenic virus pairs show that deletion of gX does not compromise efficacy of a vaccine as gI deletions do. Deletion of gX also suggests a site for replacement with antigens from other pathogens. In addition to molecular engineering of a live vaccine strain, research on PRV glycoproteins has led to the discovery that expression of the glycoprotein gp50 makes cells resistant to PRV infection. Perhaps this observation could be extrapolated to the level of a whole animal to allow engineering of pigs to become an alternative to engineered vaccines.

Animals↗
PubMed2023

Water a major source of endocrine-disrupting chemicals: An overview on the occurrence, implications on human health and bioremediation strategies.

Endocrine disrupting chemicals (EDCs) are toxic compounds that occur naturally or are the output of anthropogenic activities that negatively impact both humans and wildlife. A number of diseases are associated with these disruptors, including reproductive disorders, cardiovascular disorders, kidney disease, neurological disorders, autoimmune disorders, and cancer. Due to their integral role in pharmaceuticals and cosmetics, packaging companies, agro-industries, pesticides, and plasticizers, the scientific awareness on natural and artificial EDCs are increasing. As these xenobiotic compounds tend to bioaccumulate in body tissues and may also persist longer in the environment, the concentrations of these organic compounds may increase far from their original point of concentrations. Water remains as the major sources of how humans and animals are exposed to EDCs. However, these toxic compounds cannot be completely biodegraded nor bioremediated from the aqueous medium with conventional treatment strategies thereby requiring much more efficient strategies to combat EDC contamination. Recently, genetically engineered microorganism, genome editing, and the knowledge of protein and metabolic engineering has revolutionized the field of bioremediation thereby helping to breakdown EDCs effectively. This review shed lights on understanding the importance of aquatic mediums as a source of EDCs exposure. Furthermore, the review sheds light on the consequences of these EDCs on human health as well as highlights the importance of different remediation and bioremediation approaches. Particular attention is paid to the recent trends and perspectives in order to attain sustainable approaches to the bioremediation of EDCs. Additionally, rigorous restrictions to preclude the discharge of estrogenic chemicals into the environment should be followed in efforts to combat EDC pollution.

Animals↗
PubMed2004

[Progress on Chlorella in the area of molecular biology].

Unicellular green alga Chlorella is a kind of important eukaryotic microalga. A lot of work about Chlorella was reported in recent years with the rapid development of algal biotechnique. In the area of molecular biology, studies of Chlorella focus on the nuclear and chloroplast genome, cloning and analysis of important genes and genetic engineering using Chlorella as vector. This review reports the research progress in these aspects.

Animals↗
PubMed2001

Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA.

Driven by the needs of functional genomics, DNA engineering by homologous recombination in Escherichia coli has emerged as a major addition to existing technologies. Two alternative approaches, RecA-dependent engineering and ET recombination, allow a wide variety of DNA modifications, including some which are virtually impossible by conventional methods. These approaches do not rely on the presence of suitable restriction sites and can be used to insert, delete or substitute DNA sequences at any desired position on a target molecule. Furthermore, ET recombination can be used for direct subcloning and cloning of DNA sequences from complex mixtures, including bacterial artificial chromosomes and genomic DNA preparations. The strategies reviewed in this article are applicable to modification of DNA molecules of any size, including very large ones, and present powerful new avenues for DNA manipulation in general.

Cloning, Molecular↗
PubMed2005

Genetically engineered cytoplasmic male sterility.

Cytoplasmic male sterility, conditioned by some maternally inherited plant mitochondrial genomes, is the most expedient method to produce uniform populations of pollen-sterile plants on a commercial scale. Plant mitochondrial genomes are not currently amenable to genetic transformation, but genetic manipulation of the plastid genome allows engineering of maternally inherited traits in some species. A recent study has shown that the Acinetobacter beta-ketothiolase gene, expressed in the Nicotiana tabacum plastid, conditions maternally inherited male sterility, laying the groundwork for new approaches to control pollen fertility in crop plants.

Cytoplasm↗
PubMed2000

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome.

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Animals↗
PubMed2004

Crop improvement through modification of the plant's own genome.

Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.

Base Sequence↗
PubMed2002

[Intersections of reconstructive surgery in the area of regenerative medicine].

Based on significant progress in biotechnology, "Regenerative Medicine" is evolving as a new discipline that could significantly change current therapeutic approaches. Medicine, stem cell biology, tissue engineering, nanotechnology, genomic research, material sciences, as well as system- and process-engineering come together to answer fundamental problems of human disease, including trauma and aging. The long-term goal is to create a profound knowledge of individualized therapy originating from nonbiological organ replacement, through hybridized systems, to fully biological implants and finally to human regenerative systems in vivo. Two model tissues, including heart valves and liver tissue are discussed in the context of in vivo and in vitro regeneration technologies. Extracellular matrix regulates tissue differentiation in at least three ways, including the biochemical composition of the matrix constituents, the 3-D organization or architecture, and via mechanical forces mediated to the cells by the matrix. The in vivo extracellular matrix constitutes a biopolymer which potentially plays a permissive role for tissue differentiation.

Animals↗
PubMed2002

Dictionary-driven prokaryotic gene finding.

Gene identification, also known as gene finding or gene recognition, is among the important problems of molecular biology that have been receiving increasing attention with the advent of large scale sequencing projects. Previous strategies for solving this problem can be categorized into essentially two schools of thought: one school employs sequence composition statistics, whereas the other relies on database similarity searches. In this paper, we propose a new gene identification scheme that combines the best characteristics from each of these two schools. In particular, our method determines gene candidates among the ORFs that can be identified in a given DNA strand through the use of the Bio-Dictionary, a database of patterns that covers essentially all of the currently available sample of the natural protein sequence space. Our approach relies entirely on the use of redundant patterns as the agents on which the presence or absence of genes is predicated and does not employ any additional evidence, e.g. ribosome-binding site signals. The Bio-Dictionary Gene Finder (BDGF), the algorithm's implementation, is a single computational engine able to handle the gene identification task across distinct archaeal and bacterial genomes. The engine exhibits performance that is characterized by simultaneous very high values of sensitivity and specificity, and a high percentage of correctly predicted start sites. Using a collection of patterns derived from an old (June 2000) release of the Swiss-Prot/TrEMBL database that contained 451 602 proteins and fragments, we demonstrate our method's generality and capabilities through an extensive analysis of 17 complete archaeal and bacterial genomes. Examples of previously unreported genes are also shown and discussed in detail.

Algorithms↗
PubMedDate not supplied

How will bioinformatics influence metabolic engineering?

Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development.

Computational Biology↗
PubMed2004

Mutagenic insertion and chromosome engineering resource (MICER).

Embryonic stem cell technology revolutionized biology by providing a means to assess mammalian gene function in vivo. Although it is now routine to generate mice from embryonic stem cells, one of the principal methods used to create mutations, gene targeting, is a cumbersome process. Here we describe the indexing of 93,960 ready-made insertional targeting vectors from two libraries. 5,925 of these vectors can be used directly to inactivate genes with an average targeting efficiency of 28%. Combinations of vectors from the two libraries can be used to disrupt both alleles of a gene or engineer larger genomic changes such as deletions, duplications, translocations or inversions. These indexed vectors constitute a public resource (Mutagenic Insertion and Chromosome Engineering Resource; MICER) for high-throughput, targeted manipulation of the mouse genome.

Animals↗
PubMedDate not supplied

Chimeric restriction enzymes: what is next?

Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of Fokl (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.

DNA Restriction Enzymes↗