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Figure 6 Gene gun-mediated transfection of living mouse embryos. Day 9.5 embryos were transfected in vitro with 1 to 3 ^m gold particles coated with nuclear-localized p-galactosidase gene. One day later, the embryos were fixed and stained with X-gal. The dark dots under this low magnification are the p-galactosidase-positive cells.

into solution and rapidly ''streams'' into the nucleus of the cells (Stephen Johnston, personal communication). This observation has several implications. First, DNA is not delivered by direct particle delivery into the nucleus. Instead, the DNA is initially delivered into the cytoplasm where it engages endogenous host cell nuclear uptake machinery for translocation into the nucleus (Fig. 1B). This active uptake into the nucleus after gene gun delivery is quite different than that observed after microinjection of DNA into the cytoplasm were little uptake is observed (21-23). This difference could occur by a number of mechanisms. The first difference is related to how much DNA is introduced and how biologically relevant that amount of DNA is. For example, cytoplasmic microinjection delivers approximately 10 to 1000 femtograms of DNA in picoliter volumes (21-23). This amount of cytoplasmic DNA equals approximately 15,000 to 1,500,000 plasmids per cell. Given that each cell only has approximately 30 femto-grams of cellular DNA, it is not surprising that the nuclear uptake machinery of the cell might be overwhelmed and disabled by the delivery of whole genome amounts (or more) of injected DNA. In contrast, the gene gun typically delivers 1 particle per cell where each particle carries approximately 100 copies of plasmid (0.006 femtograms of DNA). Therefore, under this condition of delivering a low number of plasmids to the cell in amounts that normally occur during transfection, DNA uptake by the nucleus is observed, whereas when massive amounts of DNA are delivered to the cytoplasm, the system is overwhelmed and nuclear uptake of DNA is inefficient. An alternate mechanism may involve a secondary effect of using kinetic energy to deliver particles and DNA to cells. In this mechanism, the velocity of the microparticles not only punches the particle through the plasma membrane, but also the impact of the particle with the cell may shatter this complex network of the cytoskeleton (Fig. 1B). This could disrupt the ''size exclusion'' or ''sieving'' effect of the cytoskeletal network that is speculated to impede movement of DNA through the cell (Fig. 1A). If this occurs, then the delivered DNA might be more easily translocated to the nuclear pore and into the nucleus than if DNA is simply injected into the cytoplasm. Another mechanism to explain more efficient nuclear uptake after gene gun delivery involves the use of the polyamines like spermidine to protect DNA when it is loaded onto the microparticles. In this case, the polyamine that is normally found in the nucleus bound to DNA may itself act as a nuclear localization signal to increase localization of the DNA to the nuclear pore and/or to increase uptake into the nucleus through the pore. For the gene gun, nuclear uptake does not appear to require nuclear membrane breakdown during mitosis (21) because one can effectively transfect a number of postmitotic cells, including the epidermis of the skin (14) and terminally differentiated myotubes (Fig. 5). These mechanisms (and others) may act alone or in concert to produce differences in the efficiency of DNA uptake into the nucleus by various gene delivery methods. Robust head-to-head comparisons using biologically relevant amounts of DNA are needed to extract the true biology at work.

F. Efficiency of Gene Gun Delivery

Although gene guns can address cells that are refractory to cooperative vectored gene delivery, the gene gun is limited in the total number of cells that are typically transfected in each shot. In most cases, the total number of cells addressed by this technology is typically 5% to 10% of cells within 10 to 20 cell layers of a target site (14). This limited capacity to transfect many cells at a time limits the gene therapy applications for which this technology will be effective. For example, the gene gun is clearly unsuited to gene therapy that requires that most if not all cells be modified (e.g., in Duchenne muscular dystrophy) because only a small fraction of target cells can be addressed. Likewise, the gene gun is unsuitable for therapeutic applications to tissues that are difficult to expose, such as the inner structures of the brain or the lumen of the lung. In contrast, the gene gun is suitable for addressing targets in culture, targets that are at the surface of the body [e.g., the skin (13,14), the eye (17,18)], or internal organs/tissues that can be exposed surgically [e.g., the liver (13,14), the muscle (13,24), the spleen (25)].

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