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 883 records · Page 49Linked to original sources
PubMed1999

Viruses and gene silencing in plants.

Genetic engineering of virus resistance in plants may be conferred by transgenes based on sequences from the viral genome. In many instances the underlying mechanism involves the transgenically expressed proteins. However there are other examples in which the mechanism is based on RNA. It appears that this mechanism is related to post transcriptional gene silencing in transgenic plants. This gene silencing is likely to involve antisense RNA produced by the action of a host-encoded RNA dependent RNA polymerase. The natural role of this mechanism is as a genetic immune system conferring protection against viruses. There may also be a genomic role of the process reflected in RNA directed methylation of transgenes. Further understanding of this mechanism has obvious implications for virus resistance in plants. In addition the gene silencing can be used as a component of a new technology with application in functional genomics.

Gene Expression↗
PubMed2001

[The mouse model and human disease].

The mouse is an ideal model organism for studies of human disease, because mouse is physiologically very similar to human. Also, there is a large genetic reservoir of potential models of human diseases that has been generated. In addition, high-resolution genetic and physical linkage maps are now available and the sequence of mouse genome will be completed in the near future. Furthermore, the techniques necessary for the modification of mouse genome, such as transgenic and knockout techniques, and chromosome engineering methods have been established. These techniques enable us to introduce any mutations anywhere in the mouse genome. The methods for analyzing complex genetic diseases also have been developed. These advances facilitate the identification and cloning of mouse disease loci and the establishment of new models. It makes mouse the model organism of choice by academic and industrial researchers to study human diseases. In Part I of this review, we summarize the classical and modern approaches that provide the basis of establishing mouse model of human diseases. In the following parts, we will list more than 100 mouse models of human diseases. In most of these models, the mouse mutant phenotype closely resembles the human disease phenotype. These mouse models are valuable sources for the understanding of the human diseases and they can be used to develop strategies for prevention and treatment of the diseases.

Animals↗
PubMed2001

Molecular cloning, genomic positioning, promoter identification, and characterization of the novel cyclic amp-specific phosphodiesterase PDE4A10.

We describe the cloning and expression of HSPDE4A10, a novel long form splice variant of the human cAMP phosphodiesterase PDE4A gene. The 825 amino acid HSPDE4A10 contains a unique N terminus of 46 amino acids encoded by a unique 5' exon. Exon-1(4A10) lies approximately 11 kilobase pairs (kb) downstream of exon-1(4A4) and approximately 13.5 kb upstream of the PDE4A common exon 2. We identify a rat PDE4A10 ortholog and reveal a murine ortholog by nucleotide sequence database searching. PDE4A10 transcripts were detected in various human cell lines and tissues. The 5' sequence flanking exon-1(4A10) exhibited promoter activity with the minimal functional promoter region being highly conserved in the corresponding mouse genomic sequence. Transient expression of the engineered human PDE4A10 open reading frame in COS7 cells allowed detection of a 121-kDa protein in both soluble and particulate fractions. PDE4A10 was localized primarily to the perinuclear region of COS7 cells. Soluble and particulate forms exhibited similar K(m) values for cAMP hydrolysis (3-4 microM) and IC(50) values for inhibition by rolipram (50 nM) but the V(max) value of the soluble form was approximately 3-fold greater than that of the particulate form. At 55 degrees C, soluble HSPDE4A10 was more thermostable (T(0.5) = 11 min) than the particulate enzyme (T(0.5) = 5 min). HSPDE4A10 and HSPDE4A4B are shown here to be similar in size and exhibit similar maximal activities but differ with respect to sensitivity to inhibition by rolipram, thermostability, interaction with the SRC homology 3 domain of LYN, an SRC family tyrosyl kinase, and subcellular localization. We suggest that the unique N-terminal regions of PDE4A isoforms confer distinct properties upon them.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

High efficiency retroviral vectors that contain no viral coding sequences.

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences. Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene expression. However, we have now developed a series of retroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors, the intron and exon sequences from heterologous cellular or viral genes are present. When compared with the well-known MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences.

3T3 Cells↗
PubMed1995

Social and related implications of DNA fingerprinting and other new DNA technologies.

The discovery of the structure DNA at the beginning of this half of the century, and the recognition that it is virtually the universal genetic material, made it imperative for man, sooner or later, to apply this knowledge for unexpected ends. Genetic engineering, DNA fingerprinting, sequencing of whole genomes (be they of man, plants or microorganisms), or exploitation of the differences between the DNA of the male and the female (for example, of the X and the Y sperm) have thus all been historical imperatives. Today, many such predicted technologies are a reality or have come close to being so. Such DNA-based technologies have enormous social implications, discussed in this article. The impact of these technologies on different societies is likely to be different, in view of the different social conditions that prevail, and the different historical background and cultural traditions. In India, for example, the use of DNA fingerprinting could prevent the large-scale exploitation of women, and several kinds of litigation. On a global scale, the availability of this technique and its refinement to a stage where it would become virtually foolproof could act as a deterrent to certain kinds of crime. There will also be unprecedented benefits to agriculture but also new problems that will need to be faced and resolved. Wisdom demands that we prepare ourselves for a large-scale application of the new DNA technologies that are a historical imperative, understand the problems that are likely to arise, and plan to cope with them before they descend on us in full force.

Agriculture↗
PubMed2002

Germline engineering: the moral challenges.

Not only is the use of germline genetic engineering likely in the long run to be inevitable, there are also no convincing secular moral grounds to forbid this technology in principle. Instead, the general secular moral constraints on the use of germline genetic engineering are either procedural or without predetermined content. Nor can one develop a coherent distinction between eliminating disease and enhancing human abilities. At best, in secular morality one can establish the principle to proceed with care, though the invocation of the precautionary principle argues as much in favor of the development of germline genetic engineering as against its use. Because germline genetic engineering, by eliminating certain genetic defects, offers the prospect of decreasing human suffering and decreasing the use of prenatal diagnosis and abortion, there is an obligation, all else being equal, to change the human genome. In a post-modern world, humans face the challenge of directing their own evolution, although they share no common understanding of human destiny and purpose. Such understandings, though available within religious contexts, are not available to secular bioethics.

Bioethics↗
PubMed2002

Engineered gene circuits.

A central focus of postgenomic research will be to understand how cellular phenomena arise from the connectivity of genes and proteins. This connectivity generates molecular network diagrams that resemble complex electrical circuits, and a systematic understanding will require the development of a mathematical framework for describing the circuitry. From an engineering perspective, the natural path towards such a framework is the construction and analysis of the underlying submodules that constitute the network. Recent experimental advances in both sequencing and genetic engineering have made this approach feasible through the design and implementation of synthetic gene networks amenable to mathematical modelling and quantitative analysis. These developments have signalled the emergence of a gene circuit discipline, which provides a framework for predicting and evaluating the dynamics of cellular processes. Synthetic gene networks will also lead to new logical forms of cellular control, which could have important applications in functional genomics, nanotechnology, and gene and cell therapy.

Animals↗
PubMed1997

Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

Alleles↗
PubMed2005

Complete chloroplast genome sequence of Gycine max and comparative analyses with other legume genomes.

Lack of complete chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Therefore, we sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of Glycine, Lotus and Medicago confirm the organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes.

Base Sequence↗
PubMed2000

The commercial production of chemicals using pathway engineering.

Integration of metabolic pathway engineering and fermentation production technologies is necessary for the successful commercial production of chemicals. The 'toolbox' to do pathway engineering is ever expanding to enable mining of biodiversity, to maximize productivity, enhance carbon efficiency, improve product purity, expand product lines, and broaden markets. Functional genomics, proteomics, fluxomics, and physiomics are complementary to pathway engineering, and their successful applications are bound to multiply product turnover per cell, channel carbon efficiently, shrink the size of factories (i.e., reduce steel in the ground), and minimize product development cycle times to bring products to market.

Alcohols↗
PubMed2004

Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors.

Continuous protein production with baculovirus expression vectors in insect-cell bioreactors is characterized by a dramatic drop in heterologous protein production within a few weeks. This is mainly due to the spontaneous deletion of the heterologous gene(s) from the baculovirus genome and/or to the rapid accumulation of defective interfering baculoviruses (DIs). Cell culture experiments with bacmid-derived baculoviruses showed that spontaneous deletions in the foreign bacterial artificial chromosome (BAC) sequences readily occurred. These deletions correlated with a low density of baculovirus homologous (repeat) regions (hrs), which are located dispersed throughout the baculovirus genome and are believed to act as origins of viral DNA replication (oris). To test the hypothesis that deletions are more likely to occur in regions with a low ori density, the properties of bacmid-derived baculoviruses with an additional hr in the unstable BAC sequences were compared to the standard bacmid-derived baculovirus in a continuous cascaded insect-cell bioreactor configuration. All viruses were equipped with a green fluorescent protein (GFP) gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). The insertion of an extra hr in the BAC vector led to improved genetic stability of adjacent sequences, resulting in prolonged protein expression. The maintenance of the BAC sequences appeared to be dependent on the orientation of the inserted hr. The advantages of the utilization of hrs to improve the stability of baculovirus expression vectors for the large-scale protein production in insect-cell bioreactors are discussed.

Animals↗
PubMed2026

Biomaterial-Integrated Electroporation for Therapeutic Delivery: From Gene Editing to Tumor Ablation and Immune Modulation.

Electroporation has evolved from a membrane-permeabilization method into a versatile therapeutic platform for intracellular delivery, locoregional tumor intervention, and bioelectrically regulated treatment. Depending on pulse intensity and duration, electroporation operates in two distinct modes: reversible electroporation (RE), which transiently permeabilizes the plasma membrane to enable delivery of nucleic acids, proteins, and small molecules while preserving cell viability, and irreversible electroporation (IRE), which causes permanent membrane damage for non-thermal tissue ablation. Increasingly, the therapeutic scope of electroporation is being expanded through integration with biomaterials, including nanocarriers, hydrogels, soft conductors, and micro/nanoengineered bioelectronic interfaces. These material-assisted strategies improve cargo protection, field confinement, local retention, tissue conformity, and spatiotemporal control, thereby extending electroporation beyond conventional transfection toward gene editing, engineered cell manufacturing, electrochemotherapy, tumor ablation, immune modulation, and transdermal or localized delivery. In this Review, we summarize the biophysical principles of RE and IRE, discuss how biomaterials reshape electroporation performance across therapeutic settings, compare the design logic of major biomaterial-assisted electroporation platforms, and highlight key translational challenges, including pulse-material compatibility, manufacturing scalability, in vivo dosimetry, and regulatory complexity.

Humans↗
PubMed2001

High-throughput proteomics: protein expression and purification in the postgenomic world.

Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. Protein expression and purification are key processes in these studies, but have typically only been applied on a case-by-case basis to proteins of interest. Researchers are addressing the challenge of parallel expression and purification of large numbers of gene products through the principles of high-throughput screening technologies commonly used in pharmaceutical development. Some of the issues relevant to these approaches are discussed here.

Genome, Human↗
PubMed1999

Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complexes.

In order to assess efficient DNA gene transfer into human primary cell cultures derived from the skeleton we tested two viral-based procedures. First, replication-deficient recombinant adenoviruses (ADV) were used to infect post-confluent human marrow stromal fibroblasts (HMSF) and human trabecular bone (HTB) cells. Both cell types were readily infected by modified adenoviral vectors carrying a reporter gene making this virus an attractive candidate to facilitate DNA gene transfer. In a second approach we coincubated DNA with ADV that had polylysine (PLL) covalently attached. With this ADV/PLL/DNA complex, very efficient gene transfer into multilayered HMSF and HTB cell cultures was observed, and DNA coincubated with unmodified ADV failed to be effectively transferred. These data imply that the covalently bound PLL more effectively binds exogenous DNA, resulting in a highly efficient internalization event in both cell types. Thus, this latter method has many advantages over conventional ADV gene transfer procedures. It is simple, rapid, and it does not require engineering of DNA into the viral genome, thereby allowing transfer of large fragments of DNA.

Adenoviridae↗