Electro-acupuncture-mediated gene transfer

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Retroviral-mediated gene transfer

There are now many examples of the successful expression of genes transduced by retroviruses in studies from outside the field of neuroscience. Retroviruses will undoubtedly also prove to be effective tools for neuro-scientists interested in expressing cloned neurotransmitter and receptor genes. There are also other less obvious applications of retroviruses, such as their insertional mutagenic effects, which may be useful in studies of the genetic factors and biochemical mechanisms involved in, for example, neurotoxicity. Strong cellular promoters have been identified by retroviral infection and subsequent rescue of the flanking genomic DNA. Retroviruses can be employed again to reintroduce these regulatory sequences back into cells. In this way the complexities of gene expression in the many subpopulations of neurons may be unraveled. Retroviruses can also serve as very useful genetic markers in studies of development and lineage relationships. Retroviruses may be used to efficiently transfer oncogenes into neuronal cells to create new cell lines. This application exploits one of the natural traits of retroviruses--oncogenesis--which led to their original discovery. Finally, there are neurotropic retroviruses that could serve as important vectors for delivering genes into neurons. Studying these retroviruses may lead to an understanding of how they cause neuropathologic changes in the CNS.     

Horizontal gene transfer

This review explores examples of horizontal genetic transfer in eukaryotes and prokaryotes. The best understood of these involves various conserved families of transposable elements, but examples of non-transposable-element-based movement of genes or gene clusters have also been identified in prokaryotic genomes. A unifying theme is the structural and DNA-sequence homology of transposable elements from widely unrelated genomes, suggesting evolutionarily conserved mechanisms for horizontal transfer. This is reinforced by the fundamental similarity in the enzymatic mechanisms of retro viral integration (by integrases) and of transposition (by transposases). The review deals with various types of horizontal transfer, the mechanisms available for such transfer, potential barriers, and the evolutionary significance of horizontal genetic transfer.     

Adeno-associated virus-mediated gene transfer

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system.     

Liposome mediated gene transfer

Liposomes, artificial membrane vesicles, are being intensively studied for their usefulness as delivery vehicles in vitro and in vivo. Substantial progress has been made in the development of procedures for liposome preparation, targeting and delivery of contents. The broad flexibility now available in the design of the structure and composition of liposomes, coupled to recent reports of liposome mediated gene transfer in animals, suggest that liposome technology is now poised to be utilized in the creation of custom-designed cell-type-specific gene transfer vehicles.     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.     

Lateral and oblique gene transfer

Sequence information from complete genomes, and from multiple loci of strains within species, is transforming the way that we investigate the evolution of bacteria. Such large-scale assessments of bacterial genomes have provided evidence of extensive gene transfer and exchange. Except in rare cases, these two processes do not seem to be coupled: certain species, such as Escherichia coli, undergo relatively low levels of gene exchange; but the emergence of pathogenic strains is associated with the acquisition of numerous virulence factors by lateral gene transfer.     

Sperm-mediated gene transfer in mice

Sperm-mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, but the efficiency in mice has been controversial. To determine the basis of the variability of the procedure in mice, we undertook a large, collaborative study of sperm-mediated DNA transfer to mouse eggs in well-established laboratory conditions for in vitro fertilization and offspring development following embryo transfer. Sperm were incubated with plasmid DNA during the capacitation period and then added to freshly ovulated mouse oocytes for fertilization; cleaved embryos were then transferred to the oviducts of pseudopregnant recipients for gestation. From a total of 75 experiments, 13 produced 130 transgenic offspring, amounting to 7.4% of total fetuses. In five experiments, more than 85% of offspring were transgenic, but the factors leading to this high success rate were not discovered. Clustering of such a low frequency event could account for the disparate reports of transgenic success with sperm-mediated DNA transfer to mouse offspring. Discovering the factors important to success would not only allow this simplified approach to become an important tool in the generation of transgenic mice, but could also lead to important insights into natural protective mechanisms against sperm-mediated transfer of foreign DNA. Mol. Reprod. Dev. 50:406–409, 1998. © 1998 Wiley-Liss, Inc.     

Irradiation and fusion gene transfer

Irradiation and fusion gene transfer (IFGT) is a technique that spans the gap between the limitations of molecular methods and somatic-cell genetics, allowing the separation of DNA fragments between 0.25 and 30 Mb in size. In conjunction with genetic linkage analysis and physical mapping techniques, IFGT provides a very useful addition to methods for cloning disease loci, and mapping chromosomes and entire genomes.     

Polybrene/DMSO-assisted gene transfer

Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.