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PubMed2003

Ethical issues in modern biological technologies.

Today's biology-based technologies have emerged from a historical imperative and as an inevitable consequence of developments in modern biology beginning in the last half-century. They can be classified into almost 30 different areas, ranging from the use of gene therapy for human beings, enzyme engineering, stem cells and cloning, to marine biotechnology, bioinformatics, nanotechnology and biological warfare among many others. Many of them have major sociopolitico-economic, moral, ethical and legal implications. They include genetic engineering, gene therapy, tissue culture, stem cell work, the new DNA technologies, commercialization of traditional plant-based drug formulations, assisted reproduction techniques, cloning technologies, organ transplantation, bioinformatics, and biological weapons. Examples of the ethical implications of several of these items will be considered. They will be assessed with special reference to ethical implications in respect of assisted reproduction techniques, of worldwide importance today, particularly for a country such as India.

Biological Warfare↗

PubMed2004

Gene therapy approaches for cardiovascular diseases.

Cardiovascular diseases are one of the main causes of mortality in Western countries. Gene therapy is emerging as a potential strategy for the treatment of cardiovascular diseases, such as peripheral arterial disease, ischemic heart disease, restenosis after angioplasty, vascular bypass graft occlusion and transplant-associated coronary artery disease. Since the initial experiments more than one decade ago, remarkable progress has been made in the field of gene transfer and human clinical trials are underway. In here we give an overview of available gene transfer strategies describing several delivery routes and currently used vectors in animal studies and clinical trials. Hereby we want to focus on new approaches including the potential combination of gene therapy with cell therapy and tissue engineering, gene silencing and recently developed techniques for targeting genes to the vascular wall and the myocardium.

Animals↗

PubMed2003

[Gene-enhanced tissue engineering: applications in osteoinduction using cultured mesenchymal stem cells transduced with the bFGF gene].

To investigate the effect of basic fibroblast growth factor(bFGF) gene transfection on the proliferation and differentiation of mesenchymal stem cells (MSCs) and to provide basis for accelerating bone defect repairing using gene-enhanced tissue engineering technology, Rabbit periosteum-derived MSCs were transfected with the full-length rat bFGF cDNA in vitro. The transient and stable gene expression of bFGF were determined by immunohistochemistry. The proliferation and the synthesis alkaline phosphatase (ALP) and osteocalcin(OC) of the transfected MSCs were also examined. The results showed that bFGF cDNA could be transferred into osteoblasts and expressed stably at least 4 weeks. The proliferation and OC content of genetically modified MSCs were increased significantly, whereas the ALP activity remained no change. In conclusion, transfer of gene encoding bFGF to MSCs increases its proliferation and osteogenesis property. Based on the successful conjunction of the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, molecular tissue engineering, was put forward for the first time. As a new branch of tissue engineering, it represents both a new area and an important trend in tissue engineering research.

Alkaline Phosphatase↗

PubMed2006

Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer and protein delivery of BMP-2.

Bone tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs) and silk fibroin biomaterials were combined to study the impact of viral transfection on MSC osteogenic performance in vitro. MSCs were transduced with adenovirus containing a human BMP-2 (Ad-BMP-2) gene at clinically reasonable viral concentrations and cultured for 4 weeks. Controls with nontransfected MSCs, but exposed to exogenous BMP-2 concentrations on an analogous time profile as that secreted by the Ad-BMP-2 group, were compared. Both the Ad-BMP-2 MSC group and the exogenous protein BMP-2 group strongly expressed osteopontin and bone sialoprotein. Cells secreted a matrix that underwent mineralization on the silk fibroin scaffolds, forming clusters of osseous material, as determined by micro-computed tomography. The expression of osteogenic marker proteins and alkaline phosphatase was significantly higher in the Ad-BMP-2 MSC group than in the exogenous protein BMP-2 group, and no significant differences in mineralization were observed in two of the three MSC sources tested. The results demonstrate that transfection resulted in higher levels of expression of osteogenic marker genes, no change in proliferation rate and did not impact the capacity of the cells to calcify tissues on these protein scaffolds. These findings suggest additional options to control differentiation where exogenous additions of growth factors or morphogens can be replaced with transfected MSCs.

Adenoviridae↗

PubMed2006

Inheritance and state switching of genetic toggle switch in different culture growth phases.

Using the gene engineering methods, one can construct simple artificial gene networks with two stable functioning regimes (bistable genetic systems). Such genetic systems make it possible for cells with identical genotype to inherit two alternative phenotypes. The toggle switch is just one of the types of bistable genetic systems. In this work, we investigate the inheritance and switching of toggle switch functioning regimes in the cells at different culture growth phases. It is shown that during transition into the stationary growth phase the inheritance of stable states is disturbed and variations in the toggle-switching rate are more possible in different cells. Also, simultaneous expression of two genes of the system has been experimentally modelled. According to our results, the culture growth phase in this period determines later on the ratio between cell phenotypes in a population.

Escherichia coli↗

PubMed2003

Defensive applications of gene transfer technology in the face of bioterrorism: DNA-based vaccines and immune targeting.

Gene transfer involves the introduction of an engineered gene into a person's cells with the expectation that the protein expressed from the gene will produce a therapeutic benefit. Strategies based on this principle have led to the approval of > 600 clinical trials and enrollment of approximately 3500 subjects worldwide in attempts to treat diseases ranging from cancer to AIDS to cystic fibrosis. While gene therapy has met with limited success and still has many hurdles to overcome before it sees wide application, it may be useful as a defensive strategy against bioterrorism agents including infectious microbes and toxins. Although many defensive strategies are possible, immunological strategies are currently the most developed and are being actively applied to the development of strategies against several of the most virulent potential bio-weapons. While most of these strategies are not yet ready for human application, DNA-based vaccines appear to be among the most promising in the fight against bioterrorism.

Animals↗

PubMed2004

[Cloning, expression of soluble VEGFR2 fragment and its effect on tumor angiogenesis].

OBJECTIVE: To study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro. METHODS: RT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection. RESULTS: 1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control. CONCLUSION: Soluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.

Animals↗

PubMed2004

[Potential ecological risks of transgenic trees].

A new approach to genetic improvement of trees has been introduced with the birth of gene engineering technique. Compared to that in crops, gene introduction in trees has bigger potential ecological risk in environmental release and extension, because trees, most of which are wind-dispersed, grow on various habitats, have longer life span, and subject to relatively more extensive management. Extensive plantation of transgenic trees may reduce the genetic diversity of the trees concerned. Co-evolution of pests and pathogens is likely to be caused under the pressure of long-term and continuous selection of the trees derived from gene transferring. Escaping of exogenous gene may have a certain kind of influence on fitness of plants naturally generated, and as a result, have influence on species diversity in the natural world. It is not reasonable for China, a developing country, to reject gene introduction as an approach to promote forestry development. It is also important, on the other hand, to take future ecological safety into consideration because it is unwise to get present profit at the cost of future profit. To strengthen basic study on gene transferring, adopting safe management of varieties generated from gene transferring and increasing funds on research and management of transgenic trees are believed to be measures to decrease, to the greatest extent, ecological risks brought about by gene transferring of trees, and to quicken transformation of products of trees derived from gene-transferring into merchandises.

Ecosystem↗

PubMed2006

Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny.

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.

Blotting, Western↗

PubMed2002

In vitro systems for tissue engineering.

Tissue engineering, by necessity, encompasses a wide array of experimental directions and scientific disciplines. In vitro tissue engineering involves the manipulation of cells in vitro, prior to implantation into the in vivo environment. In contrast, in vivo tissue engineering relies on the body's natural ability to regenerate over non-cell-seeded biomaterials. Cells, biomaterials, and controlled incubation conditions all play important roles in the construction and use of modern in vitro systems for tissue engineering. Gene delivery is also an important factor for controlling or supporting the function of engineered cells both in vitro and post implantation, where appropriate. In this review, systems involved in the context of in vitro tissue engineering are addressed, including bioreactors, cell-seeded constructs, cell encapsulation, and gene delivery. Emphasis is placed upon investigations that are more directly linked to the treatment of clinical conditions.

Animals↗

PubMed2002

Chromosome engineering: prospects for gene therapy.

Recent advances in chromosome engineering and the potential for downstream applications in gene therapy were presented at the Artificial Chromosome Session of Genome Medicine: Gene Therapy for the Millennium in Rome, Italy in September 2001. This session concentrated primarily on the structure and function of human centromeres and the ongoing challenge of equipping human artificial chromosomes (HACs) with centromeres to ensure their mitotic stability. Advances in the 'bottom up' construction of HACs included the transfer into HT1080 cells of circular PACs containing alpha satellite DNA, and the correction of HPRT deficiency in cells using HACs. Advances in the 'top down' construction of HACs using telomere associated chromosome fragmentation in DT40 cells included the formation of HACs that are less than a megabase in size and transfer of HACs through the mouse germline. Significant progress has also been made in the use of human minichromosomes for stable trans-gene expression. While many obstacles remain towards the use of HACs for gene therapy, this session provided an optimistic outlook for future success.

Animals↗

PubMed1993

[The genetic language: grammar, semantics, evolution].

The genetic language is a collection of rules and regularities of genetic information coding for genetic texts. It is defined by alphabet, grammar, collection of punctuation marks and regulatory sites, semantics. There is a review of these general attributes of genetic language, including also the problems of synonymy and evolution. The main directions of theoretical investigations of genetic language and neighbouring questions are formulated: (1) cryptographic problems, (2) analysis of genetic texts, (3) theoretical-linguistic problems, (4) evolutionary linguistic questions. The problem of genetic language becomes one of the key ones of molecular genetics, molecular biology and gene engineering.

Codon↗

PubMed1991

[Contribution by discussion on the release of transgenic organisms. I. Genetic aspects].

Problems which might be caused by the release of transgenic organisms are discussed from the viewpoint of genetics, population genetics and ecology. On this background, the differences between "classical" methods to recombine genetic information and the more recent ones of gene engineering are pointed out and the predictability of gene interactions is discussed. Moreover, the possibility of uncontrolled reproduction of released transgenic organisms and the distribution of manipulated genes are demonstrated on the basis of our knowledge on nontransgenic organisms. Finally, the problems of risk assessment are emphasized.

Genetic Engineering↗

PubMed1991

[Contribution to the discussion on the release of transgenic organisms. II. Ecologic aspects].

Ecology↗

PubMed2006

Genetically engineered underdominance for manipulation of pest populations: a deterministic model.

We theoretically investigate the potential for introgressing a desired engineered gene into a pest population by linking the desired gene to DNA constructs that exhibit underdominance properties. Our deterministic model includes two independently segregating engineered constructs that both carry a lethal gene, but suppress each other. Only genotypes containing both or neither construct are viable. Both constructs also carry the desired gene with an independent regulatory mechanism. We examine the minimal number of individuals of an engineered strain that must be released into a natural population to successfully introgress the desired gene. We compare results for strains carrying single and multiple insertions of the constructs. When there are no fitness costs associated with the inserted constructs (when the lethal sequences are not expressed), the number of individuals that must be released decreases as the number of insertions in the genome of the released strain increases. As fitness costs increase, the number of individuals that must be released increases at a greater rate for release strains with more insertions. Under specific conditions this results in the strain with only a single insertion of each construct being the most efficient for introgressing the desired gene. We discuss practical implications of our findings.

Animals↗

PubMedDate not supplied

[Genetically engineered mutants of the envelope protein of the RNA-containing bacteriophage].

Expression of the coat protein gene of RNA bacteriophage fr in Escherichia coli cells leads to the formation of capsid-like structures of ca. 25 nm in diameter, which are immunologically indistinguishable from the native phage fr capsids. The modification strategy of the coat protein gene by gene engineering technique was developed in order to localize coat protein regions, which are exposed on the capsid surface and are capable to include foreign amino acid inserts without an appreciable effect on the capsid self-assembly. The oligonucleotide linkers, coding short amino acid sequences and bearing also convenient restriction sites, were synthesized and inserted into different regions of the coat protein gene. The mutant proteins, containing insertions of 2-12 amino acids in potentially exposed regions, were obtained. It was shown that N- and C-terminal insertions, as well as the insertion into codon 51 in the RNA-binding region, do not prevent the self-assembly. The regions (codons 96 and 112) were also revealed, insertions in them decreased drastically the protein yield as a consequence of a block in the self-assembly.

Base Sequence↗

PubMed2002

A new substrate specificity for acyl transferase domains of the ascomycin polyketide synthase in Streptomyces hygroscopicus.

Ascomycin (FK520) is a structurally complex macrolide with immunosuppressant activity produced by Streptomyces hygroscopicus. The biosynthetic origin of C12-C15 and the two methoxy groups at C13 and C15 has been unclear. It was previously shown that acetate is not incorporated into C12-C15 of the macrolactone ring. Here, the acyl transferase (AT) of domain 8 in the ascomycin polyketide synthase was replaced with heterologous ATs by double homologous recombination. When AT8 was replaced with methylmalonyl-CoA-specific AT domains, the strains produced 13-methyl-13-desmethoxyascomycin, whereas when AT8 was replaced with a malonyl-specific domain, the strains produced 13-desmethoxyascomycin. These data show that ascomycin AT8 does not use malonyl- or methylmalonyl-CoA as a substrate in its native context. Therefore, AT8 must be specific for a substrate bearing oxygen on the alpha carbon. Feeding experiments showed that [(13)C]glycerol is incorporated into C12-C15 of ascomycin, indicating that both modules 7 and 8 of the polyketide synthase use an extender unit that can be derived from glycerol. When AT6 of the 6-deoxyerythronolide B synthase gene was replaced with ascomycin AT8 and the engineered gene was expressed in Streptomyces lividans, the strain produced 6-deoxyerythronolide B and 2-demethyl-6-deoxyerythronolide B. Therefore, although neither malonyl-CoA nor methylmalonyl-CoA is a substrate for ascomycin AT8 in its native context, both are substrates in the foreign context of the 6-deoxyerythronolide B synthase. Thus, we have demonstrated a new specificity for an AT domain in the ascomycin polyketide synthase and present evidence that specificity can be affected by context.

Acyltransferases↗

PubMedDate not supplied

[Streptomycetes--producers of polyketide antibiotics].

The review is dedicated to biosynthesis of polyketides compounds by streptomycetes. It contains new data about structure of streptomycete chromosome, structure and action mechanism of polyketidesynthases of I and II types and gene clusters determining the biosynthesis of polyketides. A new promising approach is considered--the combinatorial biosynthesis of hybrid "unnatural" antibiotics by means of gene engineering construction of recombinant strains bearing the biosynthetic genes of bacterial aromatic polyketidesynthases.

Anti-Bacterial Agents↗