Microinjection

Another approach that has been explored for plastid transformation is microinjection. Microinjection into discrete plastids of intact plant cells entails the use of a syringe consisting of a submicron diameter glass capillary driven by the thermal expansion of galinstan, a liquid metal alloy 

4 DNA Transfer Mediated by T-DNA oi Agrobacterium tumefaciens and Other Approaches 

Biopharmaceuticals in Plants Toward the Next Century of Medicine 

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Microinjection is performed on single-cell embryos that are either collected following natural insemination or are produced in vitro by maturing and... 

Protease has also been demonstrated to exhibit antitumor activity. Intratumoral microinjection of proteases from Serratia marcescens into mice with sohd tumors resulted in necrosis and solubilization of the tumor mass (53). 

Blinks, J. R., et al. (1978). Practical aspects of the use of aequorin as a calcium indicator Assay, preparation, microinjection, and interpretation of signals. Method. Enzymol. 57 292-328. 

Franck, Z., Footer, M., Bretscher, A. (1990). Microinjection of villin in cultured cells induces rapid and long-lasting changes in cell morphology but does not inhibit cytokinesis, cell motility or membrane ruffling. J. Cell Biol. Ill, 2475-2485. 

Handel, S.E., Hendry, K.A.K., Sheterline, P. (1990). Microinjection of covalently cross-linked actin oligomers causes disruption of existing actin filament architecmre in PtK2 cells. J. Cell Sci, 97, 325-333. 

Sinard, J.H., Pollard, T.D. (1989). Microinjection into Acanthamoeba castellanii of monoclonal antibodies to mysoin II slows but does not stop cell locomotion. Cell Mot. Cytoskel. 12,42-52. 

Unmodified PNAs have been introduced to cells by microinjection [33], electroporation [60, 62, 76], co-transfection with partially hybridized DNA oligos [43, 61,... 

Classical gene transfer methods still in use today are diethylamino ethyl (DEAE)-dextran and calcium phosphate precipitation, electroporation, and microinjection. Introduced in 1965, DEAE-dextran transfection is one of the oldest gene transfer techniques [2]. It is based on the interaction of positive charges on the DEAE-dextran molecule with the negatively charged backbone of nucleic acids. The DNA-DEAE-dextran complexes appear to adsorb onto cell surfaces and be taken up by endocytosis. 

Much evidence supports this scheme. For example, neuronal depolarisation increases the amount of free synapsin in the cytosol and microinjection of CAM kinase II into the terminals of the squid giant axon or brain synaptosomes increases depolarisation-evoked transmitter release. By contrast, injection of dephosphorylated synapsin I into either the squid giant axon or goldfish Mauthner neurons inhibits transmitter release. 

Kask, A, Rago, L and Harro, J (1998) Anxiolytic-like effect of neuropeptide Y (NPY) and NPY13-36 microinjected into vicinity of locus coeruleus in rats. Brain Res. 788 345-348. 

One problem with both these theories is that disruption of noradrenergic transmission by selective adrenoceptor antagonists has little impact on the development of escape deficits. However, such antagonists do prevent the reversal of learned helplessness by antidepressants (reviewed by Stanford 1995). Also, it would be most unlikely that a deficit in only one neurotransmitter system fully accounts for learned helplessness. Indeed, there is plenty of evidence for a role for 5-HT in learned helplessness for instance, this behaviour is reversed by microinjection of 5-HT into the prefrontal cortex (Davis et al. 1999). Finally, it is clear that opioid, GABAergic and cholinergic systems (among others) are all linked with this behavioural deficit and even dihydropyridine antagonists of Ca + channels prevent its development. 

Cape EG and Jones, BE (1998) Differential modulation of high-frequency gamma-electroencephalogram activity and sleep-wake state by noradrenaline and serotonin microinjections into the region of cholinergic basalis neurons. J. Neurosci. 18 2653-2666. 

O Keefe, S. J., Wolfes, H., Kiessling, A. A., and Cooper, G. M. (1989). Microinjection of antisense c-mos oligonucleotides prevents meiosis II in maturing mouse eggs. Proc. Natl. Acad. Sci. USA 86 7038-7042. 

Figure 2. Role of c-mos in meiosis of mouse oocytes. Mouse oocytes microinjected with antisense c-mos oligonucleotides fail to progress from meiosis I to meiosis II, instead reforming nuclei following polar body extrusion (O Keefe et al., 1989).

An additional sequence element within the mouse ZP3 promoter has been identified as the binding site of an oocyte-specific protein called OSP-1 (Schickler et al., 1992), although the possible effect of mutations of this element on transcriptional activity has not been established. Nonetheless, it is interesting that a similar sequence (GATGA) is present 40 nucleotides upstream of the c-mos transcription initiation site (Fig. 5), although deletion of this element had no discernible effect on the activity of the c-mos promoter in microinjected oocytes (Pal et al., 1991). 

Jaenisch, R. (1985). Mammalian neural crest cells participate in normal embryonic development on microinjection into postimplantation embryos. Nature 318 181— 183. 

Elucidation of the Ras pathway in vertebrates was based on the identification of proteins having sequence homologies with those present in Drosophila and C. elegans. Expression or microinjection... 

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