Microinjection advantages

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. 

Caged compounds have the advantage of exhibiting their activities when and where we wish them to do so, in combination with microinjection. Allan et al. S3mthesized a potential photoactivatable caged ABA, the l-(2-nitro)phenylethyl ester (13), which was microinjected into guard cells [16]. ABA was released internally by UV photolysis and subsequently caused stomatal closure. This result suggests intracellular ABA perception. 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

Enucleation of unfixed oocytes under mineral oil. In order to isolate intact microinjected P-labeled RNAs from oocyte nuclei and cytoplasms, we (M.P.T.) enucleate unfixed oocytes under mineral oil. An advantage of isolating nuclei under mineral oil is that it virtually eliminates cytoplasmic and nuclear leakage that can occur when enucleations are performed under aqueous media (Paine et ai, 1983). Also, oil-isolated nuclei retain their functional properties (Lund and Paine, 1990 Paine et ai, 1992) and can be injected with RNAs to study nuclear events (Terns and Dahlberg, 1994). 

The study of lamin assembly by the microinjection of fluorescently labeled lamins has the advantage that controlled amounts of renatured protein can be injected, and that the manipulated cells can be continuously observed. The formation of heterodimeric complexes between microinjected 5-IAF-labeled lamins and the endogenous lamins can be largely excluded because the fluorescently labeled lamins are mainly in the form of homodimers assembled during dialysis. Comparison of our microinjection data with results obtained by transfection experiments demonstrates that the mutant phenotype can be obscured in transfected cells by overexpression and/or the formation of heterooligomeric complexes of mutant molecules with wild-type lamins of the transfected cells (Schmidt and Krohne, 1995). Therefore we believe that transfection experiments performed with cDNAs coding for mutated nuclear proteins should eventually be controlled by the microinjection of fluorescently labeled protein. 

There are different techniques to overcome the cell membrane barrier and introduce exogenous impermeable compounds, such as dyes, DNA, proteins, and amino acids into the ceU. Some of the methods include lipofection, fusion of cationic liposome, electroporation, microinjection, optoporation, electroinjection, and biolistics. Electroporation has the advantage of being a noncontact method for transient permeabilization of cells (Olofsson et al., 2003). In contrast to microinjection techniques for single cells and single nuclei (Capecchi, 1980), the electroporation technique can be applied to biological containers of sub-femtoliter volumes, that are less than a few micrometers in diameter. Also, it can be extremely fast and well-timed (Kinosita et al., 1988 Hibino et al., 1991), which is of importance in studying fast-reaction phenomena (Ryttsen et aL, 2000). 

Microinjection is a tool to overcome the plasma membrane permeability barrier to the introduction of charged molecules, polypeptides, or DNA plasmids into cells. In essence, microinjection treats the cell as the test tube and uses a microinjection capillary needle as the pipette to add small volumes of solution to the cytoplasm or nucleus. The approach has major advantages (i) the technique is highly synchronous with a few hundred cells being injected over 10-20 minutes, (ii) intracellular environment and cell morphology is preserved, (iii) the reaction vessel is small, that is, the size of a cell, and correspondingly reagent dilution is confined to the... 

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