Technological advances and genomics in metazoan parasites

Molecular biology has provided the means to identify parasite proteins, to define their function, patterns of expression and the means to produce them in quantity for subsequent functional analyses. Whole genome and expressed sequence tag programmes, and the parallel development of powerful bioinformatics tools, allow the execution of genome-wide between stage or species comparisons and meaningful gene-expression profiling. The latter can be undertaken with several new technologies such as DNA microarray and serial analysis of gene expression. Proteome analysis has come to the fore in recent years providing a crucial link between the gene and its protein product. RNA interference and ballistic gene transfer are exciting developments which can provide the means to precisely define the function of individual genes and, of importance in devising novel parasite control strategies, the effect that gene knockdown will have on parasite survival.     

Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers

The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency and/or restricted applicability. Activated polyamidoamine (PAMAM)-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications.     

Development and application of technology for large scale cloning of cattle

Mammalian cloning technologies originally developed as methods of testing hypotheses about the mechanisms of cell differentiation. Embryo splitting procedures demonstrated that each of the cells in the early embryo are capable of developing into a complete new individual. Nuclear transplantation technologies have shown that loss of genetic sequences or even irreversible repression of gene function are also not mechanisms of cell differentiation. Therefore, both of these methods can be used for producing genetically identical animals. Nuclear transplantation has the advantage of being able to produce unlimited numbers of identical offspring. Highly efficient procedures have been developed for nuclear transplantation in mammals and several important characteristics of donor cells have been described. Unfortunately, the efficiency of producing cloned offspring is still low and many factors affecting the development of nuclear transfer embryos to term remain to be investigated. The tremendous potential of the technology for use in agriculture and medicine, however, will ensure that these problems are addressed and solved.     

Ex vivo gene transfer and correction for cell-based therapies

Cell-based therapies are fast-growing forms of personalized medicine that make use of the steady advances in stem cell manipulation and gene transfer technologies. In this Review, I highlight the latest developments and the crucial challenges for this field, with an emphasis on haematopoietic stem cell gene therapy, which is taken as a representative example given its advanced clinical translation. New technologies for gene correction and targeted integration promise to overcome some of the main hurdles that have long prevented progress in this field. As these approaches marry with our growing capacity for genetic reprogramming of mammalian cells, they may fulfil the promise of safe and effective therapies for currently untreatable diseases.     

Nonviral gene delivery: techniques and implications for molecular medicine

Medical research continues to illuminate the origins of many human diseases. Gene therapy has been widely proposed as a novel strategy by which this knowledge can be used to deliver new and improved therapies. Viral gene transfer is relatively efficient but there are concerns relating to the use of viral vectors in humans. Conversely, nonviral vectors appear safe but inefficient. Therefore, the development of an efficient nonviral vector remains a highly desirable goal. This review focuses on the numerous challenges preventing efficient nonviral gene transfer in vivo and discusses the many technologies that have been adopted to overcome these problems.     

siRNA delivery technologies for mammalian systems

Inhibition of gene expression using the RNA interference (RNAi) pathway is rapidly becoming the method of choice for studying gene function in mammalian cells. However, successful knockdown of the target gene requires efficient delivery of short interfering RNAs (siRNAs). Several technologies have been developed that enable effective delivery of siRNAs to both cells in culture and whole animals. These technologies will allow the use of RNAi to study gene function in mammalian model systems in which classical methods are often limited and costly.     

Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization. Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the development of variety-independent and efficient gene transfer methods for most of the major agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment has resulted in variety-independent and practical generation of large numbers of independently-derived crop plants. ACCELL™ technology is currently being utilized for the routine transfer of valuable genes into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.     

多结构域酶的结构域进化关系

近年来随着生命科学新技术、新方法的涌现,酶蛋白结构和功能研究逐渐深入。具有多结构域的酶蛋白中各个结构域常具有独立的催化或结合底物的功能,在重组酶和组合生物合成研究中具有极大的研究和应用价值。这些结构域功能和组织方式的多样性,是研究分子进化的基础。对结构域进行进化分析对于研究多结构域酶的进化过程、功能相近酶之间的关系,以及对酶的分类鉴定等有重要意义。本文从结构域的重复性、结构域的水平基因转移和结构域的重组等方面出发,对多结构域酶中结构域之间进化关系的研究成果进行综述。     

Gene therapy of severe combined immunodeficiencies

The concept that the outcome of a devastating disease can be modified by inserting a transgene into abnormal cells is appealing. However, the gene-transfer technologies that are available at present have limited the success of gene therapy so far. Nevertheless, severe combined immunodeficiencies are a useful model, because gene transfer can confer a selective advantage to transduced cells. In this way, a proof of concept for gene therapy has been provided.     

High-throughput proteomics: protein expression and purification in the postgenomic world.

Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. Protein expression and purification are key processes in these studies, but have typically only been applied on a case-by-case basis to proteins of interest. Researchers are addressing the challenge of parallel expression and purification of large numbers of gene products through the principles of high-throughput screening technologies commonly used in pharmaceutical development. Some of the issues relevant to these approaches are discussed here.     

Development and application of gene therapy technologies

The success of hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency (X-SCID) was a major breakthrough in the field of gene therapy. However, two patients treated with this gene therapy developed leukemia at a later time, and retroviral vector-mediated gene transfer was considered to trigger leukemogenesis; i.e. insertional mutagenesis caused activation of LMO 2 gene, which was one step toward leukemia development. To cope with this serious problem, basic studies are required to improve the safety of retroviral vectors and to develop the method for site-specific integration of transgenes. In addition, we have to develop technologies such as selective amplifier genes (SAGs), the system for selective expansion of transduced cells, in order to obtain therapeutic efficacy of hematopoietic stem cell gene therapy in many other disorders. Moreover, clinical applications of AAV vector are promising from the standpoint of safety issue, because this vector is derived from non-pathogenic virus. AAV vector is appropriate for gene transfer into neurons, muscles, and hepatocytes. For example, gene therapy for Parkinson's disease is investigated using AAV vectors. Genetic manipulation is also one of the indispensable technologies in the field of regeneration medicine, and further promotion of basic research is important.     

The construction of transgenic and gene knockout/knockin mouse models of human disease

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.     

Systematic approaches to mouse mutagenesis

A major challenge in post-genomics is the systematic determination of mammalian gene function. A variety of mouse mutagenesis technologies, both gene- and phenotype-driven, are being used to underpin systematic and comprehensive approaches to mammalian gene function studies. Recently, a number of centres have completed large-scale ENU mutagenesis programmes that employ a phenotype-driven approach to the generation of mouse mutants. The use of ENU mutagenesis represents a powerful and efficient approach to mammalian gene-function studies, but many parallel developments are needed in downstream technologies to properly harness the new enlarged mouse-mutant resources that are being created.     

Low-volume jet injection for efficient nonviral in vivo gene transfer

The transfer of naked deoxyribonucleic acid (DNA) represents an alternative to viral and liposomal gene transfer technologies for gene therapy applications. Various procedures are employed to deliver naked DNA into the desired cells or tissues in vitro and in vivo, such as by simple needle injection, particle bombardment, in vivo electroporation or jet injection. Among the various nonviral gene delivery technologies jet injection is gaining increasing acceptance because it allows gene transfer into different tissues with deeper penetration of the applied naked DNA. The versatile hand-held Swiss jet injector uses pressurized air to force small volumes of 3 to 10 μL of naked DNA into targeted tissues. The β-galactosidase (LacZ) reporter gene construct and tumor necrosis factor α gene-expressing vectors were successfully jet injected at a pressure of 3.0 bar into xenotransplanted human tumor models of colon carcinoma. Qualitative and quantitative expression analysis of jet injected tumor tissues revealed the efficient expression of these genes in the tumors. Using this Swiss jet-injector prototype repeated jet injections of low volumes (3–10 μL) into one target tissue can easily be performed. The key parameters of in vivo jet injection such as jet injection volume, pressure, jet penetration into the tumor tissue, DNA stability have been defined for optimized nonviral gene therapy. These studies demonstrate the applicability of the jet injection technology for the efficient and simultaneous in vivo gene transfer of two different plasmid DNAs into tumors. It can be employed for nonviral gene therapy of cancer using minimal amounts of naked DNA.     

High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses

Primary neurons are a common tool for investigating gene function for survival and morphological and functional differentiation. Gene transfer techniques play an important role in this context. However, the efficacy of conventional gene transfer techniques, in particular for primary motoneurons is low so that it is not possible to distinguish whether the observed effects are representative for all neurons or only for the small subpopulation that expresses the transfected cDNA. In order to develop techniques that allow high gene transfer rates, we have optimized lentiviral-based gene transfer for cultured motoneurons by using a replication-defective viral vector system. These techniques result in transduction efficacies higher than 50%, as judged by EGFP expression under the control of SFFV or CMV promoters. Under the same conditions, survival and morphology of the cultured motoneurons was not altered, at least not when virus titers did not exceed a multiplicity of infection of 100. Under the same cell culture conditions, electroporation resulted in less than 5% transfected motoneurons and reduced survival. Therefore we consider this lentivirus-based gene transfer protocol as a suitable tool to study the effects of gene transfer on motoneuron survival, differentiation and function.     

Philosophy and practice of variety-independent gene transfer into recalcitrant crops

Summary Commercialization and marketing of agricultural products derived from recombinant DNA technologies is now becoming a reality. Genetically engineered tomatoes are poised to appear in supermarket shelves in the United States sometime next year, with other crops, including cotton, maize, soybean, rapeseed, potatoes, cucurbids, raspberries and others, scheduled for release 1 to 2 yr later (Hamilton and Ellis, 1992). One of the major breakthroughs responsible for the rapid creation of commercializable products has been the development of gene transfer methods capable of introducing foreign DNA into elite germplasm. This review examines some of the principles and applications of such gene transfer technologies in relation to conventional alternatives that are limited by cell culture, host, and genotype. Advantages and disadvantages of various gene transfer methods will be discussed. Special emphasis will be given to particle bombardment, as methods based on this technology paved the way for the engineering of a number of important agronomic species. Presented in the Session-in-Depth New Approaches to Plant transformation at the 1992 World Congress on Cell and Tissue Culture, Washington, DC, June 20–25, 1992.