Nanodiamond bullets and their biological targets

The potential of nanodiamond bullets for use in ballistic delivery of biologically active molecules was investigated. Detonation nanodiamond particles were coated with DNA, synthetic ethylene precursor, or ethylene antagonist or were labeled with fluorescent dyes and delivered into bacteria, yeast, insect cells, and plant tissues with the help of a Bio-Rad particle delivery system PDS-1000/He. Detonation nanodiamonds are both mechanically and chemically stable and can be used as DNA carriers for biolistic transformation of cells and in delivery of biologically active molecules.     

Major improvements in biolistic transformation of suspension-cultured tobacco cells

Summary Suspension cultures of the NT1 line ofNicotiana tabacum L. were used as a model system to study plant biolistic transformation, because of their uniformity, rapid growth, and ease of handling. The β-glucuronidase gene and the neomycin phosphotransferase genes were used to assay transient and stable transformation. Numerous factors were studied and optimized, such that the frequency of transformation was increased roughly 60-fold for transient transformants and 20-fold for stable transformants. Both biological parameters (the promoter used to drive gene expression, osmotic preconditioning and posbombardment handling of the cells) and physical parameters of the bombardment process (particle acceleration device and accelerator parameters) were tested. The factors that increased transformation rates the most were promoter strength, use of a helium-driven particle accelerator, and osmotic preconditioning of the cells.     

Mesoporous TiO2 nanoparticles: A new material for biolistic bombardment

To date, only solid heavy metals such as gold or tungsten have been used as DNA carriers in biolistic bombardment of algae. In this study, we show that even a metal oxide of lower density can act as a DNA carrier. We investigated the potency of size‐controlled mesoporous titanium dioxide (TiO₂) particles. Among the six tested gas pressures, TiO₂ particles best facilitated transformation of the green alga Chlamydomonas reinhardtii at 1100 psi (approximately 7.6 MPa) and 2000 psi (approximately 14 MPa). Surprisingly, a mesoporous metal oxide with a density of approximately only one‐tenth that of gold or tungsten could be effective as a DNA carrier in biolistic bombardment of a rigid cell wall‐containing alga. In addition, we found two peaks of gas pressures in the transformation ratio irrespective of whether the particles were made of gold, tungsten, or TiO₂.     

Biological projectiles (phage, yeast, bacteria) for genetic transformation of plants

Summary Bacteriophage lambda particles, yeast cells, and bacterial cells were tested as projectiles to deliver marker/reporter genes into plant cells via the biolistic process. When phage particles were complexed to tungsten or gold particles and used to bombard tobacco cells, fewer than 15 cell clusters per plate transiently expressed β-glucuronidase (GUS). Cells of wildtype Saccharomyces cerevisiae were too large to be effective projectiles, but use of a reduced-size mutant resulted in a small number of transformants. Escherichia coli cells complexed with tungsten were the most effective projectile for plant transformation. Various methods to prepare E. coli were tested to reduce particle size, improve binding of bacteria to metal particles, and/or minimize particle clumping. In maize, the number of transformants was highest when bacteria/tungsten particles were air-dried onto macrocarriers from an aqueous solution. When maize cells were bombarded with bacteria/tungsten projectiles, rates of transient gene expression (2000 per plate) and stable transformation (50 per plate) were only two- to threefold lower than when purified DNA was used. Transformation of tobacco with E. coli projectiles was improved when the bacteria were treated with a series of ethanol and ether washes, then dried into a powder. Nevertheless, tobacco transformation was still 24- (transient) and 200-fold (stable) less than when purified DNA was used. Biological projectiles can be effective for plant transformation and are advantageous because once a DNA construct is made and put into the appropriate microorganism, the need to isolate and purify DNA for the biolistic process is eliminated, which saves time and lessens DNA shear. Such projectiles may be especially well suited where high molecular weight DNA constructs are needed.     

Foreign gene delivery into monocotyledonous species

Monocotyledonous plants are generally more recalcitrant to genetic transformation than dicotyledonous species. The absence of reliable Agrobacterium-mediated transformation methods and the difficulties associated with the culture of monocotyledonous tissues in vitro are mainly responsible for this situation. Until recently, the genetic transformation of monocotyledons was essentially performed by direct transfer of DNA into regenerable protoplasts or intact cells cultured in vitro, via polyethylene glycol treatment, electroporation or particle bombardment. Since 1990, the use of particle gun technology has revolutionized the genetic engineering of monocotyledonous species, allowing transformation to be more independent of the in vitro culture requirements. Today, at least one genotype of each major monocotyledonous crop species, including cereals, can be genetically transformed.     

Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells.

Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 10(4) per plate per 0.8 micrograms DNA. Although transformation rates for the other species were low (less than 10(2) per plate per 0.8 micrograms DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.     

Biolistic transformation of arbuscular mycorrhizal fungi

Gene transfer systems have proved effective for the transformation of a range of organisms for both fundamental and applied studies. Biolistic transformation is a powerful method for the gene transfer into various organisms and tissues that have proved recalcitrant to more conventional means. For fungi, the biolistic approach is particularly effective where protoplasts are difficult to obtain and/or the organisms are difficult to culture. This is particularly applicable to arbuscular mycorrhizal (AM) fungi, being as they are obligate symbionts that can only be propagated in association with intact plants or root explants. Furthermore, these fungi are aseptate and protoplasts cannot be released. Recent advancements in gene transformation systems have enabled the use of biolistic technology to introduce foreign DNA linked to molecular markers into these fungi. In this review we discuss the development of transformation strategies for AM fungi by biolistics and highlight the areas of this technology which require further development for the stable transformation of these elusive organisms.     

草坪草生物技术研究进展

草坪草转基因方法主要有原生质体融合法,基因枪法和农杆菌介导法。抗生素（潮霉素,G418)和除草剂（bialaphos,ppt)都可用于草坪草也可能存在生态风险性。     

Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish (Danio rerio) Tissues

We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-μm microcarriers and 1 μg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 μg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described. Received August 20, 1999; accepted January 6, 2000.     

Transient gene expression assays in rose tissues using a Bio-Rad Helios® hand-held gene gun

Rose tissues of different varieties were transformed using a Bio-Rad Helios® hand-held biolistic gun. Parameters for optimum transient expression were optimized and included rose variety, flower age, tissue, gold particle size and DNA Loading ratio. Smooth flowers without thick waxy layers and young unopened actively growing flowers were found to be better suited for the transient expression assays. The DNA amounts, gold particle amounts and size etc. were not found to influence the efficiency of the transient transformation in these tissues. These studies indicate that biolistic transformation using hand-held guns can be used for successful transient expression assays in rose flower tissues. This is especially useful for a quick and easy analysis of genes and their expression before attempting stable transformation.     

Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.     

Generation of DNA double-strand breaks and inhibition of somatic embryogenesis by tungsten microparticles in wheat

Particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used to deliver foreign DNA into target cells and tissues. Some side effects of biolistic transformation have been observed but never studied in detail. Here we present evidence that intact tungsten particles can promote a breakage of phosphodiester bonds in native DNA, at a limited number of sites. A single, double-strand break appeared within almost each of the circular pUC119 molecules after a short incubation of plasmid DNA with a suspension of tungsten particles. No further DNA cutting could be induced even if the reaction rate was accelerated by increasing the concentration of tungsten in the incubation mixture. Indirect evidence indicates that similar lesions may be generated in cellular DNA of bombarded tissues. These lesions are rapidly repaired, as evidenced by increasing incorporation of labelled DNA precursors in bombarded wheat embryos. The rate of repair is, however, not high enough to restore all the genome functions. Neither germination of mature embryos nor initiation of callus tissues from immature embryos was inhibited by biolistic bombardment. Nevertheless, the frequency of formation of somatic embryos in calli derived from bombarded embryos was markedly lower than in calli derived from control embryos. Both immediate (generation of a limited number of double-strand breaks) and remote (selective inhibition of somatic embryogenesis) side effects of the biolistic process strongly suggest that biological activity of tungsten deserves special attention. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant transformation: a simple particle bombardment device based on flowing helium

We observed that flowing helium at moderate pressures accelerated DNA-coated microprojectiles to velocities suitable for penetration of cells in intact plant tissues. The flowing helium principle permitted the construction of a simple and inexpensive transformation device that was easier to use than those previously described. This device provided efficient transformation of cells in soybean seedlings and other plants.     

草坪草生物技术研究进展

概述了草坪草植株再生体系和遗传转化体系建立的方法和进展.通过愈伤组织培养、悬浮细胞培养和原生质体培养方法对草坪草的一些种已建立较为完善的植株再生体系.在建立再生体系的基础上,利用原生质体融合、农杆菌介导、基因枪和碳化硅纤维介导等转基因方法在一些草坪草种上建立了遗传转化体系并获得了有一定价值的转基因植株.最后,对草坪草转基因存在的问题和前景作了讨论.     

Biolistic transformation of animal tissue

Summary The biolistic technique transforms cells by bombardment with DNA-coated microprojectiles. It has been used to transform plants, microbes, and organelles. We adapted a standard Biolistic PDS-1000 device for use with animals and have successfully transformed tissues in live mice. The firefly luciferase gene was introduced into mouse skin and ear tissue. One day after transformation 344±74 and 1648±254 pg of luciferase were detected in skin and ear samples, respectively. Expression of the gene product was transient but detectable up to 7 days after bombardment. A further modification of the device allowed transient transformation of liver tissue in vivo. Liver contained 293±122 pg of luciferase 1 day postransformation. Expression of the gene in liver tissue was unchanged at Day 3 but declined to low levels by Day 5. This new device allowed a fourfold increase in gene expression in ear tissue extending a minimum of 14 days. This technology is applicable to a broad range of tissues and organs in situ and makes it possible to test numerous reporters and the tissue specificity of promoters. It may also be useful in protocols for somatic cell therapy. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

The efficiency of DNA strand-break repair in two fibrosarcoma tumors and in normal tissues of mice irradiated in vivo with X rays

We have used alkaline elution to study the repair of X-ray-induced DNA strand breaks in vivo in two fibrosarcoma tumors and in several normal mouse tissues after whole-body irradiation of mice with 10-12.5 Gy of X rays. Both tumors were found to repair damage significantly faster and to a greater extent than any of the normal tissues, so that by 2 hr after irradiation the level of damage in both tumors was indistinguishable from unirradiated control values. Of the normal tissues studied, liver repaired the fastest. The kinetics for the other normal tissues were essentially the same, showing an appreciable level (7-16%) of unrepaired lesions still evident after 2 hr. Even as late as 12 hr there was a significant amount of residual damage in some tissues, with testes and spleen showing the greatest level (ca. 15%). The repair kinetics for each tissue were not appropriately described by a sum of two exponentials. In contrast, previously reported data for many homogeneous mammalian cell systems in vitro and for some tissues in vivo have shown biphasic repair kinetics. This difference may be related to heterogeneity of both cell type and environment within the tissue populations used in the investigation. The faster repair of DNA strand breaks by tumor cells relative to cells from normal tissues was not readily explainable in terms of such radiobiological parameters as overall tissue oxygenation or sulfhydryl content. Rather, it appears that the degree of differentiation of the cells within the tissue population may be a major determinant of repair proficiency. Based on a model incorporating a competition between repair and fixation of sublethal lesions, these data are consistent with the idea that tumor cells may have a repair, and hence survival, advantage over normal cells in response to ionizing radiation.     

Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants

We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The -glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Act1) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration.     

Biology of human papillomavirus (HPV) infections and their role in squamous cell carcinogenesis

Current data implicating the role of human papillomavirus (HPV) infections in squamous cell carcinogenesis may be summarised as follows: animal models have shown that PVs can induce malignant transformation; HPV involvement in both benign and malignant human squamous cell tumours has been demonstrated by morphological, immunohistochemical and DNA hybridisation techniques; HPV infections in the genital tract are venereally transmitted and are associated with the same risk factors as cervical carcinoma; the natural history of cervical HPV lesions is similar to that of CIN, namely, they have the potential to develop into carcinoma in situ; malignant transformation of PV-induced lesions seems to depend on virus type and the physical state of its DNA, e.g., whether or not it is integrated in the host cell DNA; malignant transformation most probably requires synergistic actions between the PVs and chemical or physical carcinogens, or other infectious agents; genetic disposition (at least in animals) significantly contributes to malignant transformation; immunological defence mechanisms of the host are probably capable of modifying the course of PV infections (efficacy in man remains to be elucidated). Many details of the molecular mechanisms, however, still remain to be clarified. Although BPV1 is capable of transforming fibroblasts, the way that papillomaviruses transform epithelial cells is unclear. Which gene is capable of inducing the limited cell proliferation in benign lesions? No model systems exist to provide the answer nor to elucidate the progression to malignant cells and then to invasive cancer. Improved tissue culture systems for in vitro differentiation of keratinocytes should help in elucidating the biology of papillomaviruses and their interaction with cell division and differentiation.     

Using firefly luciferase to identify the transition from transient to stable expression in bombarded wheat scutellar tissue

Transformation of wheat by the biolistic procedure is unpredictable and inefficient. To gain insight into the fate of transgenes introduced into wheat scutellar tissue by particle bombardment, the expression of the firefly luciferase gene was followed using low‐light video imaging. Luciferase expression can be detected as early as 40 to 50 min after bombardment. After 48 h, the levels of transient expression in individual scutella fall rapidly, becoming undetectable 10 to 20 days later. Luciferase activity was observed to recover in a small percentage of the material and it was from this material that transformed plants, stably expressing luciferase, were recovered. We concluded that the calluses which recover luciferase expression are stably transformed and we have termed the transition period between transient and stable expression the transformation boundary. The percentage of scutella displaying luciferase activity, as measured at 30 days post‐bombardment, was judged a realistic measure of the efficiency of the transformation procedure. The results of our experiments suggest that the selection and regeneration of plants were not major factors contributing to the poor transformation efficiencies associated with biolistic transformation. The results demonstrate that luciferase can be used to assess rapidly and quantify the efficiency of the transformation procedure without the need to produce transformed plants. This will allow different procedures to be rapidly assessed and compared and should provide valuable insight into the conditions required to improve the efficiency of DNA integration and stable expression in species recalcitrant to transformation.     

Plant transformation technology

Plant transformation has its roots in the research on Agrobacterium that was being undertaken in the early 1980s. The last two decades have seen significant developments in plant transformation technology, such that a large number of transgenic crop plants have now been released for commercial production. Advances in the technology have been due to development of a range of Agrobacterium-mediated and direct DNA delivery techniques, along with appropriate tissue culture techniques for regenerating whole plants from plant cells or tissues in a large number of species. In addition, parallel developments in molecular biology have greatly extended the range of investigations to which plant transformation technology can be applied. Research in plant transformation is concentrating now not so much on the introduction of DNA into plant cells, but rather more on the problems associated with stable integration and reliable expression of the DNA once it has been integrated.