Microinjection products

The production of heterologous proteins in transgenic animals has gained much attention in the recent past. The generation of transgenic animals is most often undertaken by directly microinject-ing exogenous DNA into an egg cell. In some instances, this DNA will be stably integrated into... 

This approach includes the production and purification of antisense transcripts in vitro and then the introduction of the antisense RNA into cells by microinjection. Compared to the antisense gene approach described above, a major advantage of this method is that a much larger amount of antisense RNA can be introduced into cells. Also, antisense RNA can be injected at a specific time and can therefore result in the transient inhibition of gene expression, which can be used in studies of gene expression at a specific time within a particular window of development. However, as RNAs are extremely sensitive to nuclease degradation, the potential pharmacological uses of antisense RNA are limited. 

Figure 12.6 Plasmid nuclear localization must precede gene expression. Synchronized TC7 cells were cytoplasmically microinjected with 1000 copies of GFP-expressing plasmids (Dean et al., 1999b). After microinjection, the cells were scored for GFP expression as a function of time. The injected plasmids expressed GFP from the CMV immediate early promoter/enhancer and either contained (open circles) or lacked (closed circles) the SV40 enhancer downstream of the GFP gene. The dotted line represents the time of cell division in this population. As can be seen, plasmids without the SV40 enhancer failed to localize to the nucleus and gave no GFP expression, while plasmids containing the sequence began to express gene product within several hours of cytoplasmic injection.

Microinjection of ferrous iron (i.e. ferrous chloride) has also been shown to produce focal edema in rat brain, the degree of which is correlated with tissue levels of the lipid-peroxidation product malonyldialdehyde. Pretreatment with vitamin E (600 mg/kg intramuscularly once daily for 5 days) together with selenium (5 ppm in the drinking water) reduced the iron-induced edema and lipid peroxidation [54]. Similarly, the 21-aminosteroid U-74006F can also reduce iron-induced opening of the blood-brain barrier [53],... 

The majority of the transgenic mice produced to date have been through the microinjection route, which was used for the production of the first transgenic mouse (Isola and Gordon, 1991). In this procedure, fine glass micropipettes are used to introduce extremely small volumes of DNA solutions into the pronucleus of... 

Fig. 7.1. Production of transgenic mice by microinjection of foreign genes.

The potential value of C2 toxin as a research tool depends on the vulnerability of cells. The experimental approach is straightforward when cells are susceptible to natural poisoning, because the two components of C2 toxin can be added to the exterior of cells and the enzymatic component will find its way to the cell interior (see for example Miyake and Ohishi, 1987 Reuner et al., 1987 Zepeda eta/., 1988). The approach is more problematic when cells are resistant to natural poisoning. In theory, resistance could be due to absence of cell surface receptors, absence of a mechanism for productive internalization, or absence of an intracellular substrate, but thus far only an absence of receptors (Fritz ef al., 1995) and an absence of substrate (Aktories et al., 1986) have been described. Cells without receptors can be rendered susceptible by using techniques that produce artificial internalization (e.g., permeabilizing the cell membrane or microinjection see Muller efal., 1992). Cells that do not have substrate are permanently resistant to poisoning. 

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