Microinjection loading

Gitman, A.G., Graessmann, A., and Loyter, A. (1985b) Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells Fusion-mediated microinjection of ricin A and simian 40 DNA. Proc. Natl. Acad. Sci. USA 82, 7209-7313. 

Earlier techniques for measuring cytosolic free Ca2+ (1-2) such as the luminescent photoprotein aequorin, the absorbance dye arsenazo III and Ca2+-sensitive microelectrodes, all required microinjection or impalements, and were therefore applied mainly to giant cells. Later, photoproteins have been loaded with various reversible permeabilization procedures (3), but the largest expansion in the range of cell types in which Ca2+ signals can be quantified has come from the development of new fluorescent indicators that can be loaded using hydrolyzable esters. Currently four fluorescent indicators are used frequently quin-2, fura-2, indo-1 and fluo-3. 

A clear disadvantage of microinjection is that it is capable of loading only a relatively small number of cells. When a statistically relevant number of independent observations has to be done by evaluating junctional coupling in many cells, then other techniques are available that allow loading of many more cells. 

Therefore, the experiments were designed in a way that the enzymatic reaction could only begin in the GV. There were basically two different strategies for performing these experiments (1) multiple loading of the selected GV with different substances and (2) loading of the macromolecules into the selected GV by microinjection and addition of the substrate molecules from the external medium. Because nucleotide triphosphates are the substrate molecules for enzymes such as polymerases, the GV membranes had to be made semipermeable. Their inherent permeability toward nucleotides was too low for enzymatic reactions to be carried out in this way. 

The earliest simultaneous measurements of [Ca +Jj and force in smooth muscle were made in strips loaded by microinjection or reversible permeabiliza-tion with the bioluminescent photoprotein aequorin (Neering and Morgan, 1980 Morgan and Morgan,... 

Prepare the eggs for transfer. Remove a maximum of 20 microinjected eggs from the microdrop culture, and wash them in M2 medium. Load the eggs into an oviduct transfer pipet (Fig. 8), as described in the preliminary procedure. 

For microinjection experiments in fish, a glass needle loaded with DNA solution is used to penetrate the plasma membrane of a one-cell-stage embryo. The injection procedure is performed with the aid of a micromanipulator under a standard dissecting microscope. Once the tip of the needle has entered the cytoplasm, approximately 1-2 nL of DNA solution containing 10 to 10 DNA molecules is injected. 

Fig. 2 NP-based transgenic strategies. NP-loaded DNA may be transfected into sperm for sperm-mediated gene transfer (i), microinjected into unfertilized or fertilized oocytes (2) ormagenofected into early cleavage-stage to blastocyst-stage embryos (4). Alternatively, DNA-loaded NPs may be delivered into stem cells (5) for production of transgenic embryos by morula aggregation of blastocyst injection...

Microinjection of Proteins into Somatic Cells Needle Microinjection and Scrape Loading... 

Compared with needle microinjection, the amount of delivery varies more extensively among scrape-loaded cells. The yield of injected cells and the extent of cell damage also vary among cell types. Cell damage often manifests as the appearance of large vacuoles. 

Chemical reactions can also take place in GUVs and the synthesis of mRNA inside giant vesicles by T7 RNA polymerase was reported by Oberholzer. Giant vesicles were prepared by the electroformation method and the feasibility of the polymerization was shown after the demonstration that the membrane of some giant vesicles was permeable to nucleotides triphosphates coming from outside the GUV in the presence of ethanol. Typically, the GUVs were loaded by microinjection with the polymerase, and the DNA template necessary for the mRNA synthesis. Finally, the kinetic of the polymerization was probed as a function of time of incubation by fluorescence spectroscopy (Figure 24). 

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