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PubMed1998

[Genetic transformation of plants, recombination processes, and gene expression regulation in transgenic plants].

Recent studies in molecular biology and gene engineering were conducive to accumulation of numerous facts which help better understand the processes of plant genome functioning and follow up the effects of recombination processes in purposefully modified genome of a transformed plant on its expression. This review analyzes the major approaches to studies and results attained in plant gene engineering and plant genome imprinting.

DNA Methylation↗
PubMed1995

Expression of factor VII by muscle cells in vitro and in vivo following direct gene transfer: modelling gene therapy for haemophilia.

Direct injection of plasmid DNA into skeletal muscle has been proposed as a method of effecting somatic gene therapy. This article describes the construction and testing of a plasmid derived expression cassette believed to confer skeletal muscle specific expression. Expression constructs were designed containing the full-length cDNAs for both coagulation factor VIII and factor VII. The engineered genes were flanked by two muscle specific regulatory elements from different myosin isoforms and by an artificial polyadenylation signal sequence. In vitro transfection of C2-myoblasts led to expression of the factor VIII gene, shown by reverse transcription and polymerase chain reaction, upon differentiation of the myoblasts. The expression of the FVII construct was tested in a C2 cell culture system and also when injected directly into mouse muscle. It was found that in cell culture the level of factor VII antigen outside the cell, ie in the cell culture medium was two- to three-fold higher than inside the cell, ie in the cell lysate. This level of expression was found to continue for the duration of cell culture maintenance and a fully functional protein was produced. In vivo transfection experiments in mice showed a substantial increase in factor VII antigen compared with the background level 4-5 days after injection. An anti-human factor VII antibody was detected 7-10 days after injection. We conclude that muscle cells in vitro secrete and efficiently carry out post-translational modifications of the engineered gene product and in vivo secrete the gene product resulting in elevation of systemic levels. The data provide the basis for the use of muscle cells as an in vivo expression system for coagulation proteins in the treatment of inherited haemostatic and thrombotic disorders.

Animals↗
PubMed2004

Bone marrow stromal cell line co-transfected with IL-2 and IL-3 genes can accelerate restoration of T-cell immunity in allo-BMT mice.

BACKGROUND: After T-cell depleted allogeneic bone marrow transplantation, impaired immune reconstitution is a major cause of morbidity and mortality in the recipient. The purpose of this study was to observe the effects of the gene-engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 on the reconstitution of T-cell immunity in allo-BMT mice. METHODS: The bone marrow stromal cell line QXMSC1 was co-transfected with IL-2 and IL-3 genes using a Tet-on gene expression system. T lymphocyte subset counts per spleen were analyzed by flow cytometry. Lymphocyte proliferation response to ConA was examined to evaluate T-cell function. CDR3 spectratyping techniques were performed to evaluate TCR repertoire diversity at various time points post-transplantation. RESULTS: Gene engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 could express IL-2 and IL-3 [1,300 ng.day(-1).10(-6) cells and 1100 ng.day(-1).10(-6) cells, respectively] under the control of doxycycline. QXMSC1-IL-2 + IL-3 in combination with allogeneic bone marrow could significantly increase the counts of CD(4)(+) and CD(8)(+) T cell, 1.72 and 1.27-fold respectively at week 3 compared with TCD-BMT group (P < 0.01); make CD(4)(+)/CD(8)(+) ratio return to normal level at week 4; enhance splenocytes mitotic response to ConA (P < 0.01), and accelerate restoration of TCR repertoire diversity in the lethally irradiated mice (P < 0.05). CONCLUSION: The gene transduced stromal cell line QXMSC1-IL-2 + IL-3 is able to accelerate T-cell immunity in allo-BMT mice.

Animals↗
PubMed2003

Skeletal muscle as an artificial endocrine tissue.

Muscle has the ability to take up and express engineered genes and, because it is a post-mitotic tissue, their half-life of expression is prolonged. Although muscle is not regarded as a secretory tissue, in many cases, the gene products enter the systemic circulation. The possibility exists, therefore, of using this approach to alter levels of endocrine and paracrine factors. As a therapeutic procedure, this method has an advantage over the administration of the peptide/protein, which has a relatively short half-life and requires repeated injections. Engineered genes in plasmid or viral vectors under the control of a muscle-specific regulatory sequence may be introduced by intramuscular injection or by the introduction of transfected myoblasts. The latter is also being used in bioreactors to produce medicinal proteins/peptides in vitro as these offer some advantages over bacterial expression systems. However, for gene therapy purposes, there are still safety issues to be addressed.

Cell Transplantation↗
PubMed1989

[Interferon in the treatment of acute viral hepatitis B].

The Soviet leukocytic alpha 2-interferon obtained by gene engineering was assessed (according to the clinical and biochemical findings) for the therapeutic efficacy in acute viral hepatitis B. The drug was injected intramuscularly to patients with grave and medium-grave hepatitis during 10 days. The treatment was instituted within the first 1-5 days of jaundice. The patients with medium-grave acute viral hepatitis B manifested a beneficial therapeutic effect of alpha 2-interferon obtained by gene engineering as did the patients with the grave disease pattern before they developed the signs of liver encephalopathy.

Acute Disease↗
PubMed2006

[High-level expression of phenylalanine ammonia-lyase in Lactococcus Lactis via synthesized sequence based on bias codons].

To construct a safer and more efficient gene engineering Lactococcus Lactis for expressing phenylalaine ammonia lyase (PAL) which will be benefit for PKU therapy, pal cDNA of Parsly and synthesized sequence based on Lactococcus Lactis bias codons were recombined into two Lactococcus Lactis NICE systems. The activities of the expressed PAL were detected, and the effect of Lactococcus Lactis bias codons on the expression of exterior protein was analyzed. The results showed that the expression level of PAL was increased by using Lactococcus Lactis bias codons in both Lactococcus Lactis NICE systems. Through which several safer andmore efficient strains of the gene engineering Lactococcus Lactis were obtained.

Cloning, Molecular↗
PubMed2002

Expression and purification of recombinant bone morphogenetic protein-2 in E.coli.

OBJECTIVE: To explore the method for producing human bone morphogenetic protein-2 (hBMP-2) by gene engineering techniques. METHODS: E.coli BL21 was transformed with recombinant plasmid pYR (pBV220-hBMP-2) under different conditions, and SDS-PAGE analysis was conducted to observe the effects of the activation status and induction time of the bacterium on the target protein expression. The inclusion bodies obtained from E.coli were purified by anion exchange chromatography DEAE and molecular sieve S-300, and the recombinant protein was renatured by dialyse. RESULTS: SDS-PAGE analysis showed a conspicuous band after induction signifying a new foreign protein with relative molecular mass of approximately 13 000. After activation of the bacteria when D600 was about 0.45, most efficient expression of rhBMP-2 was achieved which reached the peak 4 h after induction with heat. Implantation of the purified recombinant hBMP-2 resulted in proliferation of mesenchymal cells and new cartilage and bone formation, as shown by histological analysis 4 weeks after implantation. CONCLUSION: hBMP-2 produced by gene engineering techniques possesses the biological capacity of ectopic bone formation.

Animals↗
PubMed2005

Reverse-engineering gene-regulatory networks using evolutionary algorithms and grid computing.

OBJECTIVE: Living organisms regulate the expression of genes using complex interactions of transcription factors, messenger RNA and active protein products. Due to their complexity, gene-regulatory networks are not fully understood.However, by building computational models it is possible to gain insight into their function and operation. METHODS: Evolutionary algorithms are used to create computational models of gene-regulatory networks based on observed microarray data. These algorithms can be computationally intensive. They will be implemented within an existing grid computing infrastructure, that has been developed for data mining purposes, and which is able to deliver the required compute power. RESULTS: We discuss how models can built achieved using distributed and grid computing technology. In particular we investigate how Condor and JavaSpaces technology is suited to the requirements of our modeling approach. CONCLUSIONS: Determining network models of gene-regulatory networks using evolutionary algorithms not only requires considerable computational power, but also a modeling formalism that can explain the underlying dynamics.

Algorithms↗
PubMed2001

Quest for antimicrobial genes to engineer disease-resistant crops.

Antimicrobial peptides are ancient mediators of the innate defenses of all species of life. These small lytic peptides are being exploited to genetically engineer disease-resistant crop plants. It is anticipated that certain (combinations of) potent antimicrobial peptides will provide agronomically relevant levels of disease control and should contribute to more sustainable agricultural practices.

Crops, Agricultural↗
PubMed2001

Construction of a xylanase-producing strain of Brevibacterium lactofermentum by stable integration of an engineered xysA gene from Streptomyces halstedii JM8.

A xylanolytic strain of Brevibacterium lactofermentum containing the Streptomyces halstedii His-tagged xysA gene was generated. The new strain contains DNA derived from S. halstedii, expresses xylanolytic activity, and was obtained by an integrative process mediated by a conjugative plasmid targeted to a dispensable chromosomal region located downstream from the essential cell division gene ftsZ. The His-tagged Xys1 enzyme was constitutively expressed under the control of the kan promoter from Tn5 and was easily purified by use of Ni-nitrilotriacetic acid-agarose. The new strain is stable for more than 200 generations, lacks any known antibiotic resistance gene, and does not need any selective pressure to maintain the integrated gene. This strategy can be used to integrate any gene into the B. lactofermentum chromosome and to maintain it stably without the use of antibiotics for selection.

Bacterial Proteins↗
PubMed1987

[Immunoenzyme demonstration of viruses and virus-specific antibodies by using conjugates based on gene engineering-produced beta-lactamase].

Enzyme immunoassays for the detection of viral antigens and virus-specific antibodies in biological samples have been described. Molecular complexes of antibodies and beta-lactamase (penicillinase) have been used as anti-specific conjugates. To synthesize the conjugate, the enzyme obtained with the aid of genetic engineering has been used. Enzyme immunoassays have been tested for the indication of the influenza virus and virus-induced specific antibodies. Enzyme immunoassays were shown to possess certain advantages (e.g., the use of simple and nontoxic substrate) along with the sensitivity identical to that of other methods, employing peroxidase-based conjugates.

Antibodies, Viral↗
PubMed2001

In vivo imaging of gene expression:.

With the ability to readily engineer genes, create knock-in and knock-out models of human disease, and replace and insert genes in clinical trials of gene therapy, it has become clear that imaging will play a critical role in these fields. Imaging is particularly helpful in recording temporal and spatial resolution of gene expression in vivo, determining vector distribution, and, ultimately, understanding endogenous gene expression during disease development. While endeavors are under way to image targets ranging from DNA to entire phenotypes in vivo, this short review focuses on in vivo imaging of gene expression with magnetic resonance and optical techniques.

Animals↗
PubMed2003

[The study of hBMP-4 gene modified tissue-engineered bone].

OBJECTIVE: Bone marrow stromal cells (bMSCs) of rabbits transferred with mammalian hBMP-4 expression plasmid were used to construct tissue-engineered bone. Gene therapy combined with tissue-engineering technique was explored to further improve osteogenesis. METHODS: pEGFP-hBMP-4 plasmid was constructed by subcloning technique. bMSCs were then transferred with either pEGFP-hBMP-4, pEGFP plasmid by lipofectamine or left uninfected in vitro. The cells from the 3 groups were combined with natural non-organic bone (NNB) to construct tissue-engineered bones, which were subcutaneously implanted into nude mice (6 implants per group) for 4 weeks. Specimens were evaluated through histological and computerized new bone formation analysis. RESULTS: pEGFP-hBMP-4 plasmid was successfully constructed. bMSCs could attach and proliferate on the surface on NNB. In vivo experiment showed that new bone formation in pEGFP-hBMP-4 group was higher than those of the control groups. CONCLUSIONS: Tissue-engineered bone using hBMP-4 gene modified bMSCs might be an ideal alternative for the repair of bone.

Animals↗
PubMed2004

Engineering ex vivo-expanded marrow stromal cells to secrete calcitonin gene-related peptide using adenoviral vector.

Calcitonin gene-related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated CGRP gene transfer into ex vivo-expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro-CGRP and AdntlacZ, adenoviral vectors containing prepro-CGRP or nuclear-targeted beta-galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral-mediated gene transfer into ex vivo-expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro- CGRP-transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro-CGRP-transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 +/- 0.4 pmol CGRP/1 x 10(6) cells/48 hours (mean +/- standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro-CGRP-transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication-deficient recombinant adenovirus can be used to gene engineer ex vivo-expanded MSCs and that high-level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell-based cell and gene therapy strategy for the treatment of cardiovascular diseases.

Adenoviridae↗
PubMed2005

[Gene studies and nobel prize].

Gene is a DNA sequence which can be expressed and produces gene products (protein or RNA). By 2003, there are 51 Nobel Prize owners related to gene studies. Among them, 44 persons are in physiology or medicine (account for 24.72% of total 178), 7 persons are in chemistry (account for 5.69% of total 123). The paper reviews them in following 6 aspects: Drosophlie melanogaster is a good material for gene study; the double helix model of DNA structure provides a hard foundation in gene study; the studies on gene regulation illuminate many functions of gene; genetic central dogma researches created 11 Noble Prize laureates; gene engineering technologies make possible to modify and use genes; and the thorough studies of gene characteristic made us easier to understand many life phenomena.

Animals↗
PubMed2003

[Progress in the pathway engineering of ethanol fermentation from xylose utilising recombinant Saccharomyces cerevisiae].

Pathway engineering was the third generation of gene engineering. Its main goals were to change metabolic flux and open a new metabolic pathway in organism. Application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway distributions and rates. Ethanol is the most advanced liquid fuel because it is environmentally friendly. Enhancing fuel ethanol production will require developing lower-cost feedstock, and only lignocellulosic feedstock is available in sufficient quantities to substitute for corn starch. Xylose is the major pentose found in lignocellulosic materials and after glucose the most abundant sugar available in nature. Recently a lot of attentions have been focused on designing metabolic pathway of Saccharomyces cerevisiae in order to expand the substrate of ethanol fermentation, because it is a traditional ethanol producing strain and has wonderful properties for ethanol industry. However, it can not utilize xylose but convert the isomer, xylulose. Many attempts are based on introducing the genes in the pathway of xylose metabolism. The further research includes overexpressing the key enzyme or decreasing the unimportant flux. The sugars in lignocellulose hydrolyzates, therefore, could be efficiently utilized. Here, we describe the ethanol pathway engineering progress in ethanol fermentation from xylose with recombinant Saccharomyces cerevisiae.

Biotechnology↗