From structure and functions of steroidogenic enzymes to new technologies of gene engineering

This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.     

A transgenic class I antigen is recognized as self and functions as a restriction element

The function of a transgenic Dd class I molecule in the induction of immunologic tolerance to major histocompatibility complex antigens and in directing major histocompatibility complex restriction in C57BL/6 mice were investigated. All of the transgenic Dd mouse strains were found to be tolerant for the Dd antigen. Spleen cells from transgenic mice were immunocompetent but consistently failed to generate an anti-Dd cytotoxic T lymphocyte response in vitro, and skin grafts between transgenic Dd mice were not rejected. These data suggests that the Dd antigen was recognized as a self molecule. In addition, the transgenic Dd mice generated antigen-specific Dd-restricted cytotoxic T lymphocyte, indicating that the Dd antigen also functioned as a restriction element for antigen recognition. These observations demonstrate the usefulness of the transgenic mouse system for studying class I antigen expression and function.     

Prospects for using genetic transformation for improved SIT and new biocontrol methods

The genetic manipulation of non-drosophilid insect species is possible by the creation of recombinant DNA constructs that can be integrated into host genomes by several transposon-based vector systems. This technology will allow the development and testing of a variety of systems that can improve existing biological control methods, and the development of new highly efficient methods. For programs such as sterile insect technique (SIT), transgenic strains may include fluorescent protein marker genes for detection of released insects, and conditional gene expression systems that will result in male sterility and female lethality for genetic sexing. Conditional expression systems include the yeast GAL4 system and the bacterial Tet-off and Tet-on systems that can, respectively, negatively or positively regulate expression of genes for lethality or sterility depending on a dietary source of tetracycline. Importantly, strains for male sterility must also incorporate an effective system for genetic sexing, since typically, surviving females would remain fertile. Models for the use of these expression systems and associated genetic material come from studies in Drosophila and, while many of these systems should be transferable to other insects, continued research will be necessary in insects of interest to clone genes, optimize germ-line transformation, and perform vector stability studies and risk assessment for their release as transgenic strains.     

Use of recombinant Salmonella strains for supplying the macro-organism with biologically active molecules

The characterization of known Salmonella vaccine strains and different attenuated mutants used for developing new vaccines is presented. The use of attenuated Salmonella strains as vaccine vector for the supply of heterologous antigens opens prospects for the creation of effective and commonly available vaccines which approximate the "ideal" vaccine in their qualitative characteristics. The possibility of the genetic modification of attenuated strains permits their targeted reconstruction, considering the specific features of the formation of immune response to the definite heterologous antigen supplied to the body by the bacterial vector.     

Antibodies generated from human immunoglobulin miniloci in transgenic mice.

One approach to the production of human monoclonal antibodies focusses on the creation of transgenic mice bearing human immunoglobulin gene miniloci. Whilst such loci undergo lymphoid-specific gene rearrangement, only a small proportion of mouse B cells express the human immunoglobulin chains; the miniloci thus contribute poorly to serum immunoglobulin. Attributing this poor performance to competition between the transgenic and endogenous immunoglobulin loci, we crossed mice bearing a human immunoglobulin heavy-chain (HulgH) minilocus with animals that had been rendered B cell-deficient by disruption of their endogenous heavy-chain locus. The results were dramatic: the human minilocus rescued B cell differentiation such that effectively all B cells now expressed human mu chains. The concentration of antibody in the mouse serum recognised by anti-human mu increased to a concentration about one sixth that in human serum. The HulgH antibodies are heterogenous with diversity being generated by both combinatorial and junctional processes. Following antigen challenge, specific antibody is elicited but at low titre.     

Setting Objectives and Defining Outputs for Ecological Restoration and Habitat Creation

The past 40 years have seen the legal and policy framework for nature conservation in Britain extend from protection and preservation to include enhancement through techniques such as ecological restoration and habitat creation. Clear objectives need to be set for ecological restoration and habitat creation schemes because the processes involve human intervention in combination with natural factors operating over time. Objectives are required for both management and monitoring in order to enable measurement of success or failure. The most effective way to achieve high-quality restoration and creation schemes is to define the output of the process—a habitat, vegetation type, or biological community. The better the definition of the output, the greater the need to define the inputs and the nature of the intervening processes. Ecologists and environmental managers have a key role to play in establishing the degree of definition necessary in order to achieve a particular objective.     

Induction of carbohydrate-specific antibodies in HLA-DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use.

Over the last few years, anticancer immunotherapy has emerged as a new exciting area for controlling tumors. In particular, vaccination using synthetic tumor-associated antigens (TAA), such as carbohydrate antigens hold promise for generating a specific antitumor response by targeting the immune system to cancer cells. However, development of synthetic vaccines for human use is hampered by the extreme polymorphism of human leukocyte-associated antigens (HLA). In order to stimulate a T-cell dependent anticarbohydrate response, and to bypass the HLA polymorphism of the human population, we designed and synthesized a glycopeptide vaccine containing a cluster of a carbohydrate TAA B-cell epitope (Tn antigen: alpha-GalNAc-Ser) covalently linked to peptides corresponding to the Pan DR 'universal' T-helper epitope (PADRE) and to a cytotoxic T lymphocyte (CTL) epitope from the carcinoembryonic antigen (CEA). The immunogenicity of the construct was evaluated in outbred mice as well as in HLA transgenic mice (HLA-DR1, and HLA-DR4). A strong T-cell dependent antibody response specific for the Tn antigen was elicited in both outbred and HLA transgenic mice. The antibodies induced by the glycopeptide construct efficiently recognized a human tumor cell line underlying the biological relevance of the response. The rational design and synthesis of the glycopeptide construct presented herein, together with its efficacy to induce antibodies specific for native tumor carbohydrate antigens, demonstrate the potential of a such synthetic molecule as an anticancer vaccine candidate for human use.     

Monitoring foreign gene incorporation into the plastome of Chlamydomonas reinhardtii by multiplex qPCR

The genetic material of the Chlamydomonas reinhardtii chloroplast can be easily manipulated and creation of transgenic plastomes is of interest for both photosynthetic research and for biofuel and biomass production. Because multiple copies of the chloroplast genome are present, it is important to understand whether, following the introduction of a foreign gene, the resulting transgenic plastome is homoplasmic or heteroplasmic. By quantitative PCR together with a simple DNA extraction procedure and a series of DNA oligonucleotides the following protocol will determine the extent of foreign gene incorporation into a host chloroplast plastome. This approach is used to follow the degree of heteroplasmy following biolistic transformation of several transgenic strains. The approach used is quick, simple to set up, and gives an accurate quantitation of foreign genes within of the chloroplast plastome. Possible future uses of the technique are discussed.     

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

Abstract To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.     

Analysis of Tumor Suppressor Genes Using Transgenic Mice

Identification and analysis of tumor suppressor genes has relied chiefly upon studies of human sporadic tumors and of tumors harvested from familial cancer syndrome patients. One methodology that is proving to be extremely useful both in analyzing the function of these genes and in identifying new tumor suppressor genes involves the creation of transgenic mice that contain targeted mutations that functionally inactivate tumor suppressor genes. Studies using such mice have provided insight into the role of tumor suppressor genes in cell growth and in embryonic development. The creation of mice that harbor mutations in one or both alleles of a targeted gene has permitted anin vivoanalysis of the tumor suppressing properties of the gene and facilitated investigation of cell cycle control and differentiation of multiple cell lineages within the organism. Sophistication of gene targeting techniques will likely result in the creation of more lines of mice bearing genetic modifications in tumor suppressor genes, permitting an even finer detailed analysis of tumor suppressor gene functions.     

Molecular approaches for identification and construction of novel insecticidal genes for crop protection

Summary The insecticidal cry (crystal) genes from Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. Discovery of new insecticidal genes is of importance for delaying the development of resistance in target insects. The diversity of Bt strains facilitates isolation of new types of cry and vip (vegetative insecticidal protein) genes. PCR is a useful technique for quick and simultaneous screening of Bt strains for classification and prediction of insecticidal activities. PCR together with other methods of analysis such as RFLP, gene sequence determination, electrophoretic, immunological and chromatographic analysis of Cry proteins and insect bioassays for evaluation of toxicity have been employed for identification of new insecticidal proteins. Some other new approaches have also been devised. Many Bt strains with novel insecticidal genes have been found. A desired combination of Cry proteins can be assembled via site-specific recombination vectors into a recipient Bt strain to create a genetically improved biopesticide. For better pest control, the cry genes have been transferred to plants. Stacking of more than one insecticidal gene is required for resistance management in transgenic crops. Modification of Cry proteins through protein engineering for increasing the toxicity and/or the insecticidal spectrum is also a promising approach, but requires detailed understanding of the structure and function of these proteins and analysis of toxin-receptor interactions. More research into this area will provide useful insights for the design of toxins for management of insect resistance. Insecticidal genes from other bacteria and plants are also being examined for their potential for deployment in transgenic crops. Stringent implementation of resistance management is needed for maintaining the efficacy of Bt transgenic crops and deriving maximum economic and environmental benefit.     

Murine model of allergen induced asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world (1). More than 8% of the US population has asthma, with the prevalence increasing (2). As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals (3). Availability of a variety of transgenic strains further increases the attractiveness of these models (4). Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma (5). The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models (5,6), and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive (7) and non-invasive (8) techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.     

Antibody discovery: the use of transgenic mice to generate human monoclonal antibodies for therapeutics

Technical advances made in the 1980s and early 1990s resulted in monoclonal antibodies that are now approved for human therapy. Novel transgenic mouse strains provide a powerful technology platform for creating fully human monoclonal antibodies as therapeutics; ten such antibodies have entered clinical trials since 1998 and more are in preclinical testing. Improved transgenic mouse strains provide a powerful technology platform for creating human therapeutics in the future.     

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.     

CRISPR/Cas9介导靶向敲除拟南芥BRI1突变体的鉴定

以拟南芥(Arabidopsis thaliana)油菜素内酯受体BRI1为目的基因,利用CRISPR/Cas9基因编辑技术定向编辑拟南芥BRI1,以期获得更多BRI1的突变体,为后续BRI1功能的进一步深入研究奠定基础。通过筛选转基因植株,对编辑后的BRI1进行测序分析,结果显示该突变体中BRI1基因序列由于新碱基的插入导致提前终止。同BRI1强突变体bri1-710一样,相比于野生型对照均对BL处理不敏感,但相比于bri1-710,该突变体植株较大,暗示BRI1 N端可能在BR信号途径中有重要作用。因此该研究可为后续进一步研究拟南芥及其他同源物种的BRI1功能提供可靠的参考依据。     

Expression of heterologous genes in plant systems: New possibilities

The basic methods used in current practice for stable and transient expression of heterologous genes in plants are presented and compared. The key areas of research in the heterologous expression of genes in plants have been identified by analyzing literature and experimental data: modeling of metabolic pathways; creation of marker-free transgenic plants; the search for new regulatory elements and plant genes influencing the efficiency of expression of heterologous genes in plants; development of new methods for analyzing of transgenic plants and new approaches to the expression of heterologous genes in plants.     

Aggregation Behavior in Wildtype and Transgenic Zebrafish

Recent advances in the development and availability of genetically modified animals enable researchers to examine the effects of phenotypic characters on social behavior. In fish, shoaling behavior is known to be influenced by characteristics such as body coloration, striping pattern, body shape, and size. GloFish^TM are genetically engineered zebrafish (Danio rerio) that express red fluorescent protein (RFP), resulting in on overall red coloration under the dark longitudinal stripes. The GloFish pattern is distinct from the light body coloration underlying the dark longitudinal stripes seen in wildtype zebrafish. We presented wildtype and transgenic RFP zebrafish with same‐sex shoals of both strains of fish in dichotomous choice tests. No preference for either of the shoals was shown, however, both strains showed significant preferences for swimming near shoals vs. swimming near an empty tank compartment. When presented with opposite‐sex individuals of both strains, no preference was shown by either sex of either strain. Thus, the red body coloration of transgenic zebrafish does not appear to affect choice of social partner, in either a shoaling or a potentially reproductive context.     

Effects of experimental warming on wild and transgenic oriental fruit fly,Bactrocera dorsalis

Climate change may influence the application efficiency of transgenic marking, such as in mark–release–recapture (MRR) experiments or sterile insect technique (SIT). Wild and transgenic fruit flies of Bactrocera dorsalis were subjected to oscillating regimes that represent current temperature conditions (mean: 28.6°C) and various future possible scenarios (means: 30.0, 32.5 and 35.0°C). As the temperature was increased to 30.0°C, the negative effects on adult fecundity and demographic parameters (net reproductive rate and intrinsic rate of increase) of only the transgenic cohorts increased. With a moderate warming (32.5°C), negative effects were observed on the net reproductive rate for both fly strains, and these effects on the life‐history traits (adult fecundity and longevity) and intrinsic rate of increase were stronger in the transgenic than in the wild cohorts, with reference to the trait values at 30.0°C. A severe warming (35.0°C) resulted in the failure of all individuals of both fly strains to reach adulthood. We suggest parametrical adjustments or decreased differences in fitness with refined transgenesis under current and future climate conditions, which can reduce the marking limitations of pest management and eradication programmes.