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PubMed1992

Genetically engineered antibodies: progress and prospects.

Techniques of genetic engineering and expression have been applied to the production of antibodies in a variety of expression systems. Novel antibodies have been produced with a variety of modifications: as chimeric antibodies, as "humanized" antibodies, with catalytic groups, as bifunctional or fusion proteins, and as functional fragments such as Fabs or Fvs. The domain structure of the antibody is favorable to such manipulation; the novel proteins often retain their antibody-derived activity and acquire new properties as well. Chimeric and complementarity-determining region (CDR)-grafted antibodies have been effective in immunotherapy, but problems of immunogenicity remain. Combinatorial libraries produced in bacteriophage may present an alternative to animal immunization as a source of antigen-binding specificities. Structural and mutational analysis of variable regions is providing useful information about the requirements of the variable region for antigen binding. Careful analysis and comparison of effector functions among immunoglobulin isotypes may be applied to the design of effective therapeutic antibodies.

Animals↗

PubMed1985

Genetic engineering of laboratory and livestock mammals.

Recent advances in recombinant genetics have made possible the transfer of cloned genes from one organism to the genome of another. Research with mice made transgenic by insertion of rat or human genes has provided direct evidence that transferred genes can be incorporated into the germline and expressed in the recipient. Current technology for gene transfer involves microinjection of the recombinant genes into the male pronucleus of the zygote. Resulting transgenic mice, when mated as adults, produced offspring that contained and expressed the transgenes. These observations serve as indications of the possibilities that exist for genetic engineering in livestock species. Although there are some technical problems to be overcome before livestock embryos can be genetically altered by these means, the genes for producing growth hormone transgenic livestock are currently available, and research groups are working toward this objective. In addition to this work with growth hormone genes, there are many other potential applications for genetic engineering livestock to produce more highly efficient production; however, there is considerable research to be done before the full potential of this technology can be achieved. It will be necessary to identify other genes that have potential for improving the production efficiency of livestock, and it will be necessary to gain a more complete understanding of the developmental and molecular biology of livestock. The potential impact of this technology in farm animal production is enormous, but, in the short term, it will be a costly endeavor.

Animal Husbandry↗

PubMed1999

Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol.

Ethanol is an effective, environmentally friendly, nonfossil, transportation biofuel that produces far less pollution than gasoline. Furthermore, ethanol can be produced from plentiful, domestically available, renewable, cellulosic biomass. However, cellulosic biomass contains two major sugars, glucose and xylose, and a major obstacle in this process is that Saccharomyces yeasts, traditionally used and still the only microorganisms currently used for large scale industrial production of ethanol from glucose, are unable to ferment xylose to ethanol. This makes the use of these safest, most effective Saccharomyces yeasts for conversion of biomass to ethanol economically unfeasible. Since 1980, scientists worldwide have actively been trying to develop genetically engineered Saccharomyces yeasts to ferment xylose. In 1993, we achieved a historic breakthrough to succeed in the development of the first genetically engineered Saccharomyces yeasts that can effectively ferment both glucose and xylose to ethanol. This was accomplished by carefully redesigning the yeast metabolic pathway for fermenting xylose to ethanol, including cloning three xylose-metabolizing genes, modifying the genetic systems controlling gene expression, changing the dynamics of the carbon flow, etc. As a result, our recombinant yeasts not only can effectively ferment both glucose and xylose to ethanol when these sugars are present separately in the medium, but also can effectively coferment both glucose and xylose present in the same medium simultaneously to ethanol. This has made it possible because we have genetically engineered the Saccharomyces yeasts as such that they are able to overcome some of the natural barrier present in all microorganisms, such as the synthesis of the xylose metabolizing enzymes not to be affected by the presence of glucose and by the absence of xylose in the medium. This first generation of genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts relies on the presence of a high-copy-number 2 mu-based plasmid that contains the three cloned genetically modified xylose-metabolizing genes to provide the xylose-metabolizing capability. In 1995, we achieved another breakthrough by creating the super-stable genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts which contain multiple copies of the same three xylose-metabolizing genes stably integrated on the yeast chromosome. This is another critical development which has made it possible for the genetically engineered yeasts to be effective for cofermenting glucose and xylose by continuous fermentation. It is widely believed that the successful development of the stable glucose-xylose-cofermenting Saccharomyces yeasts has made the biomass-to-ethanol technology a step much closer to commercialization. In this paper, we present an overview of our rationales and strategies as well as our methods and approaches that led to the ingenious design and successful development of our genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose to biofuel ethanol.

Biomass↗

PubMed1997

Practical development of genetically engineered animals as human disease models.

Since transgenic (Tg) mice were first produced in 1980, the technology to produce genetically engineered animals, such as Tg and knock-out mice, has advanced exponentially. These animals have contributed greatly to basic research at the molecular level and have proven to be powerful tools for elucidating complex biological processes. The effort that is required to develop and establish genetically engineered animals as models for human diseases or toxicologic studies is not always appreciated by researchers and laboratory animal scientists. Genetically engineered animals remain only candidates of defined animal models until they are developed for practical use, in accordance with their objectives, through cooperation between the researchers performing the experiments and laboratory animal scientists. The practical development of these animals consists of three steps: establishment as standardized laboratory animals through the setting of quality standards and the establishment of a production and supply system; establishment as defined animal models through confirmation of the usefulness and limitations of the animal in the purpose of use; and establishment of an in vivo experimentation system (an animal experimentation system), using the defined animal models. This report serves as an introduction to those that follow and describes our concept of how best to establish genetically engineered animals as valid animal models. The development of Tg mice carrying the poliovirus receptor gene (PVR) will be used as an example. The TgPVR mice were produced by the introduction of the human poliovirus receptor gene into the mouse genome, mainly to study the molecular mechanisms of pathogenesis of the virus. The value of these mice is that they mimic human and nonhuman primate susceptibility to poliovirus infection and thus serve as a model for the study of the disease and for the assessment of poliovirus vaccines. They are being touted as a replacement for nonhuman primates in the neurovirulence testing of oral poliovirus vaccine.

Animals↗

PubMed2006

Genetic engineering neural stem cell modified by lentivirus for repair of spinal cord injury in rats.

OBJECTIVE: To explore the feasibility for therapy of spinal cord injury (SCI) by genetic engineering neural stem cell (NSC) modified by lentiviral vector. METHODS: Following the construction of the genetic engineering NSC modified by lentivirus to secrete both neurotrophic factor-3 (NT-3) and green fluorescence protein (GFP), hemisection of spinal cord at the level of T10 was performed in 56 adult Wistar rats that were randomly divided into 4 groups (n = 14), namely 3 therapeutic groups and 1 control group. The therapeutic groups were dealed with NSC, genetic engineering NSC, and concentrated lentiviral supernatant which carries both GFP and NT-3, respectively. Then used fluorescence microscope to detect the transgenic expression in vitro and in vivo, migration of the grafted cells in vivo, and used the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test to assess the recovery of function. RESULTS: The transplanted cells could survive for long time in vivo and migrate for long distance. The stable transgenic expression could be detected in vivo. The hindlimb function of the injured rats in 3 therapeutic groups, especially those dealed with genetic engineering NSC, improved obviously. CONCLUSION: It is feasible to combine NSC with lentivirus for the repair of SCI. NSC modified by lentivirus to deliver NT-3, acting as a source of neurotrophic factors and function cell in vivo, has the potential to participate in spinal cord repair.

Animals↗

PubMed2006

[Relief effect of beta-galactosidase genetically engineered lactococcus lactis on the cell toxicity caused by lactose].

OBJECTIVE: To assess the relief effect of beta-galactosidase genetically engineered Lactococcus lactis on the cell toxicity caused by lactose in vitro. METHODS: An in vitro toxic Caco-2 cell model caused by lactose was established to evaluate the relief effect of beta-galactosidase genetically engineered Lactococcus lactis. Cell morphological parameters and proliferation activity parameter were used. RESULTS: The in vitro toxic Caco-2 cell model caused by lactose was successfully established; the genetically engineered Lactococcus lactis constructed in the authors' laboratory could enable the Caco-2 cell to have normal appearance with the presence of lactose and could improve the proliferation activity with the presence of high concentration of lactose (P < 0.01). CONCLUSION: The beta-galactosidase genetically engineered Lactococcus lactis has significant relief effect on the cell toxicity caused by lactose in vitro, which lays a foundation for food-grade alternation of this bacterium.

Caco-2 Cells↗

PubMed2006

High efficiency site-specific genetic engineering of the mosquito genome.

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.

Aedes↗

PubMed1995

Genetically engineered mice: tools to understand craniofacial development.

In this review, we provide a survey of the experimental approaches used to generate genetically engineered mice. Two specific examples are presented that demonstrate the applicability of these approaches to craniofacial development. In the first, a promoter analysis of the Msx2 gene is presented which illustrates the cis regulatory interactions that defined cell-specific gene expression. In the second, a mouse model of the human disease craniosynostosis, Boston type, has been created by misregulation of the Msx2 gene product. Finally. we present a formulary of spontaneously occurring and genetically engineered mice that exhibit defects in developmental processes affecting the craniofacial complex. The purpose of this review is to provide insight into the experimental approaches that are used to create genetically engineered mice and to impress upon the reader that genetically engineered mice are well-suited to address fundamental questions pertaining to the development maintenance, and regeneration of tissues and organs.

Animals↗

PubMed1996

Microencapsulated genetically engineered live E. coli DH5 cells administered orally to maintain normal plasma urea level in uremic rats.

Safety concerns about introducing genetically engineered cells into the body have prevented their use in medical treatments. To solve this problem, we prepared polymeric membrane artificial cells (semipermeable microcapsules) containing genetically engineered live cells from the bacteria Escherichia coli DH5. When given orally, the cells remain at all times in the microcapsules and are finally excreted in the stool. During their passage through the intestine, small molecules like urea diffuse rapidly into the microcapsules and are acted on by the genetically engineered cells. This lowers the high plasma urea level to normal in uremic rats with induced kidney failure, and has exciting implications for the use of this and many other types of genetically engineered cells in a number of medical applications.

Administration, Oral↗

PubMed1993

Cytogenetic effects of promutagens in genetically engineered V79 Chinese hamster cells expressing cytochromes P450.

V79 Chinese hamster cell lines genetically engineered to express rat CYP2B1, CYP1A1, CYP1A2, and their parental cell lines V79-MZ, without acetyltransferase, and V79-NH, with acetyltransferase, were studied for chromosome aberrations and sister chromatid exchange induced by aflatoxin B1, cyclophosphamide, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The parental V79 cell lines did not show clastogenic effects. Significant clastogenic effects were observed after an 18 h exposure to aflatoxin B1 and cyclophosphamide in CYP2B1 expressing cells, to benzo[a]pyrene in CYP1A1 and CYP1A2 expressing cells, to 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine in cells, expressing CYP1A2 with or without acetyltransferase, and to cyclophosphamide in cells expressing both CYP1A2 and acetyltransferase. A significant sister chromatid exchange inducing effect was found after a 24 h exposure in each of the genetically engineered cell lines, except for benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in CYP2B1 expressing cells, and for benzo[a]pyrene in cells expressing both CYP1A2 and acetyltransferase. Thus, a battery of cell lines genetically engineered for metabolic competence may serve as a tool for investigating chromosomal changes induced by activated xenobiotics.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1989

Seeding of intravascular stents with genetically engineered endothelial cells.

The use of intravascular stents may be limited by both local thrombosis and restenosis due to intimal proliferation. In an effort to provide solutions to these problems, we seeded stents with genetically engineered endothelial cells in vitro. Using retroviral-mediated gene transfer, we inserted the gene for either bacterial beta-galactosidase or human tissue-type plasminogen activator (t-PA) into cultured sheep endothelial cells. The endothelial cells were seeded onto stainless steel stents and grown until the stents were covered. Expression of intracellular beta-galactosidase and high level secretion of t-PA were demonstrated both before and after the transduced cells were seeded onto the stents. Eight stents were expanded by in vitro balloon inflation, with observation of the seeded endothelial layer both prior to and after expansion. Most of the endothelial cells remained on the stents after balloon inflation. We conclude that intravascular stents can be coated with a layer of genetically engineered endothelial cells that can be either specifically labeled or made to secrete high levels of a therapeutic protein. Much of the layer of genetically engineered cells remains after the expansion of the stent in vitro. In vivo implantation of stents coated with genetically engineered endothelial cells may allow 1) introduction of genetically engineered endothelial cells directly into the vascular wall and 2) improvement of stent function through localized delivery of anticoagulant, thrombolytic, or antiproliferative molecules.

Animals↗

PubMed1989

Prospects for correction of thalassemia by genetic engineering.

The thalassemias are diverse genetic disorders characterized by abnormal synthesis rates of one or more proteins constituting hemoglobin (globin-chains). In the beta-thalassemias, genes encoding the beta-globin chain are intact but are abnormally transcribed or, less often, translated. In the alpha-thalassemias, genes encoding the alpha-globin chain are often deleted; abnormal transcription can also occur. The human beta- and alpha-globin genes were molecularly cloned. This review considers attempts to introduce these genes in mammalian cells by physical techniques such as chromosome transfer, transfection, fusion, micro-injection, electroporation and homologous recombination or by using DNA or RNA viruses, such as retroviruses. Currently, inefficient gene expression in host cells and the need for precise cognate regulation of globin gene expression are the major limitations to applying genetic engineering to thalassemia.

Gene Expression Regulation↗

PubMed2004

[Subcloning of human neurotrophin-3 gene and construction of its genetically engineered cell model].

OBJECTIVE: To subclone human neurotrophin-3 gene (NT3) and transfer this gene into human bone marrow mesenchymal stem cells (BM-MSCs) to construct genetically engineered cells that produce NT3 in vitro. METHODS: Human BM-MSCs were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum and 10 ng/ml epidermal growth factor. Flow cytometry (FCM) was used to examine the phenotypes of the cells. The eukaryotic expression vector pcDNA3.1(+)/NT3 was constructed and transferred into human BM-MSCs in vitro via liposomes. The genetically engineered BM-MSCs were selected several times with G418 and the clones were obtained and then amplified, followed by extraction of the RNA for detection of NT3 gene expression by reverse transcriptional (RT) PCR. The biological activity of the genetically engineered cells was examined by the collecting the supernatant of the culture medium for incubation of guinea pig cochlea hair cells. RESULTS: The cultured cells expressed CD13, CD29 and CD59, but no7 CD11, CD14, CD31, CD34, CD45, CD80, CD86, CD117 or HLA-DR. The BM-MSCs genetically modified with pcDNA3.1(+)/NT3 not only expressed and produced NT3, but also promoted the survival of the guinea pig cochlea hair cells in vitro. CONCLUSION: It is possible to construct the genetically engineered BM-MSCs that excrete NT3 in vitro.

Animals↗

PubMed1992

Genetic engineering strategies for environmental applications.

Environmental applications of genetically engineered microorganisms are currently hampered not only by legal regulations restricting their release, but also by the frequent dearth of adequate genetic tools for their construction in the laboratory. Recent approaches to strain development include the use of non-antibiotic markers as selection determinants, the use of transposon-vectors for the permanent acquisition of recombinant genes, and the utilization of expression devices based on promoters from promiscuous plasmids and biodegradative pathway genes.

Biotechnology↗

PubMed1990

Genetically Engineered Erwinia carotovora: Survival, Intraspecific Competition, and Effects upon Selected Bacterial Genera.

Environmental use of genetically engineered microorganisms has raised concerns about potential ecological impact. This research evaluated the survival, competitiveness, and effects upon selected bacterial genera of wild-type and genetically engineered Erwinia carotovora subsp. carotovora to ascertain if differences between the wild-type and genetically engineered strains exist in soil microcosms. The engineered strain contained a chromosomally inserted gene for kanamycin resistance. No significant differences in survival in nonsterile soil over 2 months or in the competitiveness of either strain were observed when the strains were added concurrently to microcosms. For reasons that remain unclear, the engineered strain did survive longer in sterilized soil. The effects of both strains on total bacteria, Pseudomonas and Staphylococcus strains, and actinomycetes were observed. While some apparent differences were observed, they were not statistically significant. A better understanding of the microbial ecology of engineered bacteria, especially pathogens genetically altered for use as biological control agents, is essential before commercial applications can be accomplished.

Journal Article↗

PubMed1995

German politics of genetic engineering and its deconstruction.

Policy-making, as exemplified by biotechnology policy, can be understood as an attempt to manage a field of discursivity, to construct regularity in a dispersed multitude of combinable elements. Following this perspective of politics as a textual process, the paper interprets the politicization of genetic engineering in Germany as a defence of the political as a regime of heterogeneity, as a field of 'dissensus' rather than 'consensus', and a rejection of the idea that the framing of technological transformation is an autonomous process. From its beginning in the early 1970s, genetic engineering was symbolically entrenched as a key technology of the future, and as an integral element of the German politics of modernization. Attempts by new social movements and the Green Party to displace the egalitarian imaginary of democratic discourse into the politics of genetic engineering were construed by the political élites as an attack on the political order of post-World War II Germany. The 1990 Genetic Engineering Law attempted a closure of this controversy. But it is precisely the homogenizing idiom of this 'settlement' which continues to nourish the social movements and their radical challenge to the definitions and codings of the politics of genetic engineering.

Genetic Engineering↗

PubMed1991

Improvement of Rhizobium inoculants by mutation, genetic engineering and formulation.

The use of mutation and genetic engineering techniques have resulted in Rhizobium strains with improved characteristics. The latter approach can provide improvement and new traits not achievable previously. Enhanced commercial Rhizobium inoculants, however, still rely solely on traditional approaches including formulation improvement, mutation, and strain selection. The lack of contribution to the commercial product lines by genetic engineering is mostly due to the time delay and financial burdens cast by the regulatory policies rather than a lack of progress in the application of modern genetic technologies. Such constraints may lessen in time as the public becomes more educated about the technologies and the subsequent political pressure decreases.

Journal Article↗

PubMed1995

Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma.

Genetically engineered viruses and viral genes inserted into retroviral vectors are increasingly being considered for experimental therapy of brain tumors. A primary target of these viruses and vectors is human gliomas, the most frequently occurring primary human brain tumor. To investigate the potential of genetically engineered herpes simplex viruses (HSVs) in the therapy of these tumors, we compared the attributes of two viruses, a recombinant from which the gamma 1(34.5) gene had been deleted (R3616) and a recombinant in which the gamma 1(34.5) gene had been interrupted by a stop codon (R4009). Previous studies have shown that these recombinants were completely devoid of the ability to multiply in the central nervous system of rodents. To pursue these studies, we developed a scid mouse glioma model. Tumor cell response (survival) for 10(3), 10(4), and 10(5) implanted MT539MG glioma cells was 38, 23, and 15 days, respectively. The results were as follows: (i) both R3616 and R4009 replicate and cause cytolysis in diverse glioma cell lines of murine and human origin in vitro, and (ii) Winn-type assays 10(5) MT539MG cells coinoculated with R3616 or R4009 as compared to saline significantly prolonged survival in a dose-dependent fashion. Mice that received only tumor cells or the wild-type parent strain of the recombinants, HSV-1(F), died within 15 days. Survival was greatest with R4009. These experiments define both a model for screening oncolytic viruses and a genetically engineered virus of significant potential use as an oncolytic agent.

Animals↗