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PubMed1995

Genetically engineered animal models of human neurodegenerative diseases.

The influence of a single gene, engineered to be normally or abnormally expressed, can be evaluated in vivo through the development of transgenic animals. Application of this approach in the study of human neurological problems is contributing to an increased understanding of the pathogenic components operative in a variety of disorders. These include Alzheimer's disease, prion encephalopathies, motor neuron disease such as amyotrophic lateral sclerosis, and fragile X syndrome, as well as a number of viral-mediated neurological disorders. These transgenic animals can also serve as models to investigate the possible involvement of additional genetic and environmental factors on the disease state. Moreover, transgenic animals can be used in the development of intervention strategies. The application of this powerful and increasingly popular tool to investigate neurodegenerative disorders is reviewed.

Amyotrophic Lateral Sclerosis↗

PubMed2006

Evolving sensitivity.

Engineering gene circuits with novel functions holds promise for broad applications in biology, engineering, and medicine. Directed evolution complements rational design as an important strategy for optimizing gene circuits and circuit elements.

Directed Molecular Evolution↗

PubMed2005

Allo-specific T-cells encoding for viral IL-10 exert strong immunomodulatory effects in vitro but fail to prevent graft rejection.

Recently, we demonstrated the capacity of allo-specific gene-engineered T lymphocytes as transport vehicle for therapeutic transgenes into allografts. In this study, the influence of viral IL-10 as therapeutic transgene was addressed. Lewis rat T-cell lines specific for DA rat alloantigens were engineered to express vIL-10 by using a retroviral gene expression system. Like T regulatory 1 cells, vIL-10 transgenic T lymphocytes express the phenotype CD4(+)25(+) and secrete, in addition to vIL-10, rat IL-10 and IFN-gamma but no IL-4. First, the capacity of vIL-10 transgenic T-cell lines to modulate alloantigen-specific immune responses was evaluated in vitro. In comparison to control MLR with no transgenic cells or equal numbers of control T(EGFP)-lymphocytes, the proliferation as well as production of IFN-gamma by naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, T(vIL-10)-lymphocytes were not able, either alone or in combination with suboptimal doses of Cyclosporine A, to prolong the survival of either DA rat cardiac or renal allografts in Lewis rat recipients. These data demonstrate that intra-graft IL-10 over-expression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.

Animals↗

PubMed2004

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae.

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast traditionally used in ethanol production from hexose. However, recombinant S. cerevisiae created in several laboratories have used xylose oxidatively rather than in the fermentative manner that this yeast metabolizes glucose. To understand the differences between glucose and engineered xylose metabolic networks, we performed a flux balance analysis (FBA) and calculated extreme pathways using a stoichiometric model that describes the biochemistry of yeast cell growth. FBA predicted that the ethanol yield from xylose exhibits a maximum under oxygen-limited conditions, and a fermentation experiment confirmed this finding. Fermentation results were largely consistent with in silico phenotypes based on calculated extreme pathways, which displayed several phases of metabolic phenotype with respect to oxygen availability from anaerobic to aerobic conditions. However, in contrast to the model prediction, xylitol production continued even after the optimum aeration level for ethanol production was attained. These results suggest that oxygen (or some other electron accepting system) is required to resolve the redox imbalance caused by cofactor difference between xylose reductase and xylitol dehydrogenase, and that other factors limit glycolytic flux when xylose is the sole carbon source.

Cell Proliferation↗

PubMed2000

Tailoring new gene delivery designs for specific targets.

Advancing biotechnology spurs the development of new pharmaceutically engineered gene delivery vehicles with the capability to target specific cell types. The low density lipoprotein (LDL) element of the terplex gene carrier is shown to be efficient in delivery to smooth muscle cells as well as inducing minimal toxicity to A7R5 cells in culture. The terplex system condensed plasmid DNA into polyplex sizes ranging from 100 to 400 nm. Terplex demonstrated higher transfection efficacy than Lipofectin. Lactose was conjugated through a poly(ethyl glycol) (PEG) spacer to poly(L-lysine) (PLL) to tailor a gene carrier capable of condensing plasmid DNA. This cationic polymer targeted Hep G2 cells preferentially in culture. The inclusion of a lactose targeting moiety greatly increased the efficacy of the gene carrier over PEG-PLL and Lipofectin. A biodegradable nanoparticle gene carrier, polysaccharide-graft-PLL and poly(D,L-lactic acid), was synthesized and characterized. The nanoparticle size was controllable by changing the copolymer concentration, where the particles could be as small as 60 nm. When polysaccharide was used in the copolymer, the nanoparticle became more DNA adsorbent. The JLI mAb was linked to PLL. This gene carrier targets the antigen confined to stage II immature cortical thymocytes for leukemia therapy. A 20,000 mw PLL backbone was sufficient for compaction of DNA. Folic acid linked to PLL through a PEG spacer is currently being studied for DNA condensation and delivery to membrane associated folate binding protein positive endothelial cancer cells.

Endocytosis↗

PubMed2005

The selective cause of an ancient adaptation.

Phylogenetic analysis reveals that the use of nicotinamide adenine dinucleotide phosphate (NADP) by prokaryotic isocitrate dehydrogenase (IDH) arose around the time eukaryotic mitochondria first appeared, about 3.5 billion years ago. We replaced the wild-type gene that encodes the NADP-dependent IDH of Escherichia coli with an engineered gene that possesses the ancestral NAD-dependent phenotype. The engineered enzyme is disfavored during competition for acetate. The selection intensifies in genetic backgrounds where other sources of reduced NADP have been removed. A survey of sequenced prokaryotic genomes reveals that those genomes that encode isocitrate lyase, which is essential for growth on acetate, always have an NADP-dependent IDH. Those with only an NAD-dependent IDH never have isocitrate lyase. Hence, the NADP dependence of prokaryotic IDH is an ancient adaptation to anabolic demand for reduced NADP during growth on acetate.

3-Isopropylmalate Dehydrogenase↗

PubMed2003

Total biosynthesis of hydrocortisone from a simple carbon source in yeast.

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Animals↗

PubMed2005

Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells defined by multilineage potential, ease to gene modification, and immunosuppressive ability, thus holding promise for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which, however, does not compensate for the very low percentage in their niches given the vast numbers of cells required for the relative studies. Taking into consideration the lack of a uniform approach for MSC isolation and expansion, we attempted in this study, by comparing various culture conditions, to identify the optimal protocol for the large-scale production of MSCs while maintaining their multilineage and immunosuppressive capacities. Our data indicate that, apart from the quality of fetal calf serum, other culture parameters, including basal medium, glucose concentration, stable glutamine, bone marrow mononuclear cell plating density, MSC passaging density, and plastic surface quality, affect the final outcome. Furthermore, the use of basic fibroblast growth factor (bFGF), the most common growth supplement in MSC culture media, greatly increases the proliferation rate but also upregulates HLA-class I and induces low HLA-DR expression. However, not only does this upregulation not elicit significant in vitro allogeneic T cell responses, but also bFGF-cultured MSCs exhibit enhanced in vivo immunosuppressive potential. Besides, addition of bFGF affects MSC multilineage differentiation capacity, favoring differentiation toward the osteogenic lineage and limiting neurogenic potential. In conclusion, in this report we define the optimal culture conditions for the successful isolation and expansion of human MSCs in high numbers for subsequent cellular therapeutic approaches.

Cell Culture Techniques↗

PubMed2003

[Expression of chimeric anti-CD3 IgG antibody in mammalian cells and analysis of its biological activity].

The anti-CD3 antibody can improve success rate of organs transplant. HIT3a, a mouse anti-CD3 antibody, was chimerized by using gene engineering methods to decrease its immunogenity. The anti-CD3 genes, heavy chain and light chain, were cloned using PCR from the vector pCANTAB 5E containing anti-CD3 scFv gene fragment, and two PCR fragments were recombined into the expression vector pKN100 with human antibody light constant domain and pG1D105 with human antibody heavy constant domain, respectively. The two vectors were co-transfected into CHO cells using liposome. The anti-CD3 antibody was detected by ELISA and Western blot assay in supernatant of transfected CHO cells culture. The primary results of competitive assays by FACS showed that anti-CD3 antibody could partially block the sites through which parent antibody (HIT3a) bind to CD3+ Jurkat cells. The result of 3H-TdR incorporation showed that the chimeric anti-CD3 antibody could stimulated proliferation of peripheral blood mononuclear cells (PBMC) as the parent antibody. In this thesis, the results of some experiments indicated that the chimeric anti-CD3 antibody expressed in CHO cells was an antibody with native biological activity, and it is possible to apply to in clinic in the future.

Animals↗

PubMed1996

[Immunohistochemical detection of antigen of human cytomegalovirus in hepatocyte].

The expression plasmid of human cytomegalovirus (HCMV) 52kd glycoprotein was induced and amplified, and the recombinant protein was extracted. The rabbit antiserum to the recombinant protein was prepared. The antiserum-reacted to the antigen of HCMV AD165, and the titer of antiserum was 1 : 3200 by using 125I-Protein A. Using Avidin-Biotinperoxidase complex technique (ABC method), we detected the HCMV 52 kd glycoprotein in the hapatic slides of 9 cases. Among them, one case (an infant died of congenital heart disease) was held as a negative control. The other 8 cases (patients with clinically diagnosed cytomegalic inclusion disease) had viral hepatitis histopathologically. Of these 8 cases, 7 had positive signals but found in the nucleus and cytomembrane of the hepatocytes, no positive signal was found in the hepatocytes of the control. The result showed that HCMV 52kd glycoprotein was located in the nucleus and on the membrane of the infected hepatocyte. The antibody prepared with the antigen of gene engineering could find the virus antigen and represent the virus gene state which was active or silent. ABC method is not only simple, sensitive and specific, but also effective in localization. It is of importance to clinical diagnosis, differential diagnosis and prognostication.

Cytomegalovirus↗

PubMed2005

Model-driven designs of an oscillating gene network.

The current rapid expansion of biological knowledge offers a great opportunity to rationally engineer biological systems that respond to signals such as light and chemical inducers by producing specific proteins. Turning on and off the production of proteins on demand holds great promise for creating significant biotechnological and biomedical applications. With successful stories already registered, the challenge still lies with rationally engineering gene regulatory networks which, like electronic circuits, sense inputs and generate desired outputs. From the literature, we have found kinetic and thermodynamic information describing the molecular components and interactions of the transcriptionally repressing lac, tet, and ara operons. Connecting these components in a model gene network, we determine how to change the kinetic parameters to make this normally nonperiodic system one which has well-defined oscillations. Simulating the designed lac-tet-ara gene network using a hybrid stochastic-discrete and stochastic-continuous algorithm, we seek to elucidate the relationship between the strength and type of specific connections in the gene network and the oscillatory nature of the protein product. Modeling the molecular components of the gene network allows the simulation to capture the dynamics of the real biological system. Analyzing the effect of modifications at this level provides the ability to predict how changes to experimental systems will alter the network behavior, while saving the time and expense of trial and error experimental modifications.

Animals↗

PubMedDate not supplied

[Comparative study of physico-chemical and biological properties of human somatotropin produced by genetic engineering and isolated from the pituitary gland].

Human somatotropin hormono (STH), produced by means of gene engineering in the complex program "Human growth hormone", managed by the Academy of Sciences of the USSR, Ministry of Medical and Biological Industry of the USSR and Ministry of Public Health of the USSR, was shown to be similar in its physico-chemical properties to the main isoform of highly purified STH, isolated from human hypophysis. As distinct from the hypophyseal STH (STHhyp) containing minor isoforms of the hormone, the preparation of biosynthetic STH (des-Phe1-STH; STHbio) proved to be homogeneous. Studies of biological properties showed that STHbio exhibited high, similar to STHhyp, immunological, growth-stimulating and insulin-like activities as well as it possessed the lipotropic effect in vivo. The lipotropic effect of STHbio in vivo was less prolonged as compared with that of STHhyp, while in vitro it was only slightly expressed in isolated rabbit fat tissue. The effect did not depend on the hormone dose, apparently due to either absence of the hormone modified forms in the STHbio preparation or other hypophyseal contaminating substances responsible for the lipotropic activity. STHbio, similarly to STHhyp, did not stimulate DNA synthesis in blood serum-free culture of human fibroblasts. Studies of STHbio biological properties suggest that multifunctionality of native STHhyp appear to depend on intrinsic specificity of its molecule.

Adipose Tissue↗

PubMed2005

Breakthrough in chloroplast genetic engineering of agronomically important crops.

Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using 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 major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using species-specific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development.

Crops, Agricultural↗

PubMed1989

Transgenic mammals and biotechnology.

Biotechnology has begun to realize the enormous potential of transgenic technology: mice with human genes that produce human proteins of therapeutic value in their milk, pigs that express bovine genes that help them gain weight and lose backfat, animals with engineered gene defects that mimic human genetic diseases.

Animals↗

PubMed1994

[Susceptibility to antimicrobial drugs of strains of bacilli used as a basis for various probiotics].

Five strains of the genus Bacillus used as components of biological preparations i.e. bacterin SL, biosporin, bactisubtil and subalin were tested for their susceptibility to 38 antimicrobial agents, The strains were simultaneously resistant to 5 antibiotics: astreonam, colistin, penicillin, ceftizoxime and cefuroxime. High susceptibility of the strains to some penicillins, cephalosporins and aminoglycosides was recorded. The gene engineered strain B. subtilis 2335(105) marked by the kanamycin resistance gene showed cross resistance to amikacin and tobramycin. Trimetoprim, sulfotrimetoprim and norfloxacin proved to be active against the strains.

Anti-Bacterial Agents↗

PubMed2003

Hardware (DNA) circuits.

A scheme is presented whereby a new genetic control circuit can be introduced into an organism, permitting the experimenter to turn the expression of a given gene (or set of genes) on or off at will. The proposed scheme involves a positive feedback loop--here, a positive regulator, the CII protein of phage lambda, with its structural gene engineered so as to require CII for its expression. This feedback loop creates the possibility of two stable steady states, with gene cII ON or OFF. Genes added downstream of cII and lacking a promoter will follow the same expression as cII. Two additional circuits allow the experimenter to switch at will between the ON and OFF states.

Bacteriophage lambda↗

PubMed2002

Gene technology and tissue engineering.

The interest in gene therapy to treat human diseases has increased with the advances in recombinant DNA technology and the improved physical, chemical and biological methods of delivering genes to mammalian cells. Areas of therapeutic interest for gene therapy relevant for tissue engineering are, for example, in the treatment of wounds, skin diseases, nerve, bone, and muscle diseases. The transfer of a gene into a cell can lead to the addition or modification of a function and may be an attractive alternative to the pharmacological use of proteins. The complementation of defective functions could also be an effective treatment for inherited skin diseases with a gene defect. The two major challenges facing gene technology in tissue engineering are the problem of identifying appropriate genes that are effective in tissue repair, and the reliable expression of the therapeutic gene at clinically beneficial levels. This review discusses principles and methods of delivering genes encoding growth factors into cells, together with their respective advantages and disadvantages.

Journal Article↗

PubMed2003

Stem cells as platforms for delivery of genes to enhance cartilage repair.

BACKGROUND: The long-held axiom put forth by Hunter in 1743, that cartilage once injured is incapable of healing, has been challenged by the technique of autologous chondrocyte transplantation. This conceptual change in the way in which orthopaedists are approaching the problem of cartilage repair has spawned a myriad of new and innovative treatment modalities. This review will focus on the new techniques and directions that our facility and other investigators are exploring to restore functional articular cartilage. METHODS: To show the usefulness and effectiveness of local tissue-engineered gene therapy, we transduced periosteal stem cells known to have osteochondral potential with either bone morphogenetic protein-7 (BMP-7) or sonic hedgehog (Shh) gene. These cells were cultured to increase the number of cells and then were seeded onto bioresorbable polymer scaffolds. Full-thickness osteochondral defects were created in the mid-trochlear region of eighty New Zealand White rabbits, and the implants containing the transduced cells were placed in the defects. Animals were killed at six, eight, twelve, and twenty-six weeks postoperatively and were examined macroscopically and histologically. RESULTS: Periosteal-derived cambium-layer cells proliferated rapidly and were easily used for transfection of both the bone morphogenic protein-7 (BMP-7) and sonic hedgehog (Shh) genes. The control defects became filled with a mixture of fibrous and fibrocartilaginous tissue. The addition of either the BMP-7 or the Shh gene significantly enhanced the quality of the repair tissue, resulting in a much smoother surface and more hyaline-appearing cartilage. There was, however, a noticeable difference in the persistence of the cartilage phase between the group that received the Shh gene and the group that received the BMP-7 gene, with the subchondral compartment in the latter group seeming to remodel with bone much faster. CONCLUSION AND CLINICAL RELEVANCE: The results of these experiments clearly demonstrate the utility of tissue-engineering strategies in which gene therapy is used to locally influence the repair environment. It is interesting to note the relative differences in the two different gene responses with regard to skeletal development and the repair process. These differences could be related to the genes' temporal patterns in skeletal development.

Animals↗