Microinjection technique

For research purposes, PET is attractive because it has relatively high spatial resolution. It must be realized, however, that, even with PET, we are not at the level of cells and molecules that is provided by the microelectrodes and microinjection techniques used in basic sleep research. So a gap still remains. We tolerate this gap because PET tells us things that would take decades (or centuries) to realize if we could use these microinjection techniques in humans (which we can t), and because we know that it is just a matter of time before the gap is closed in animals. 

Armyworm caterpillars that have been dipped in a solution of the compound will feed normally when offered untreated leaves. Using microinjection techniques, a small amount of solution of 24,055 was placed inside the mouth cavity of the armyworms they fed normally. Injection of the material into the body cavity of the caterpillars also had no effect on their feeding. Thus, feeding seems to be affected only if the insect actually bites and/or tastes the material on its food. 

Kotsanis, N. and J. Iliopoulou-Georgudaki. Arsenic induced liver hyperplasia and kidney fibrosis in rainbow trout Oncorhynchus mykiss) by microinjection technique a sensitive animal bioassay for environmental metal-toxicity. Bull. Environ. Contam. Toxicol. 62 169—178, 1999. 

By using a microinjection technique in guinea pigs, we also determined the pharmacological properties of an area producing cough-Uke responses when it was electrically stimulated. The injection of a small amount of 5-hydroxytryptamine (5-HT), atropine, and DL-2-amino-5-phosphonopentanoic acid inhibited cough responses caused by mechanical stimulation of the mucosa of the tracheal bifurcation. Since... 

P. Bucher, A. Fischer, P. L. Luisi, T. Oberholzer, P. Walde, Giant vesicles as biochemical compartments the use of microinjection techniques. Langmuir, 1998, 14, 2712-2721. 

We thank Dr. Deborah Lewis for instruction in the microinjection technique. Dr. Alex Chiu for the use of die microinjection apparatus. Dr. Miyake Katsuya for assistance with image collection. Dr. David Munn for suggesting the use of the SK-MEL-28 cell line. Dr. Andrew Hayhurst for scFv 147, and Dr. David Bickel for statistical advice. We acknowledge the Medical College of Georgia Imagh Core Facility, Medical Illustration and Photography, and Office of Biostatistics and Bioinformatics for their services. This work was... 

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). 

The microinjection technique described by Gottschalk, Morel and Mylle (5) was applied with some minor modifications. Known volumes (10 to 25 nl) of a colored solution of buffered saline containing tritiated inulin and urate labeled with 14c were injected with a calibrated pipette into proximal or distal surface convolutions. Inulin was used as the reference substance (6) and was totally recovered in the urine of the punctured kidney after technically satisfactory microinjections. It was assumed that the injected inulin remained solely within the lumen of the tubule, i.e. the liaihinal membrane was impeinneable to inulin. It was further assumed that labeling of urate did not impair its physiological properties and that excreted 14c represented urate. 

One of the main reasons that GVs are so attractive for studying is their use as microreactors. In such systems the progress of enzymatic reactions in a compartmentalized system can be studied in situ and in real time (see also chapter 22). An early example of such a system is the digestion of microinjected DNA by pancreatic DNase I in a GV This system also shows the feasibility of this microinjection technique in GVs, because it demonstrates that multiple puncturing of a target GV is possible. 

Microinjections into such spherical GVs are possible and, in most cases, these punctured vesicles remain stable over a long period and do not lose their injected material. This work shows that macromolecules such as nucleic acids and proteins can be entrapped by microinjection and that the detection limit with the nucleic acid dye YO-PRO-1 is in the range 10-50fg. With RNAs (mixture of tRNAs or midi variant RNA [13]) this limit is about 10 times higher (A. Fischer and T. Oberholzer, unpublished observation). A limitation of the present microinjection technique is the fact that the vesicles, in which a liquid has been injected, become less stable because of osmotic effects. Therefore, the injections of highly concentrated salt solutions have to be avoided. Such an injection would immediately lead to shrinkage (maybe it does grow, and then the membrane bursts) of the vesicle in such a way that it cannot be observed by this microscopy technique. 

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