Capillary microinjection

Ansorge, W. (1982). Improved system for capillary microinjection into living cells. Exp. Cell Res. 140,31-37. 

Computer-Automated Capillary Microinjection of Macromolecules into Living Cells... 

FIGURE 1 (A) Scheme of the setup of the automated capillary microinjection system (AIS). 

W. Ansorge et al. Performance of an Automated System for Capillary Microinjection into Living Cells. J. Bloc hem. Biophys. Methods 16 (1988) 283-292. 

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... 

Microinjection involves the direct injection of nucleic acids into the nucleus or cytoplasm of target cells and is the simplest approach for gene delivery. Thin glass capillaries are used to inject nanoliters of nucleic acid solution into cells. One major drawback of this method is obviously the throughput every single cell has to be manipulated individually. This limits the use of microinjection to applications in which individual cell manipulation is possible, such as genetic engineering of... 

Figure 5.2. Schematic diagram of a standard liquid chromatograph modified for use with a packed capillary column. Typical pump settings are 300-400 p,l / min with flow splitting of 1 2000 to give a column flow of 150-200 nl / min. The microinjection valve has a 40 nl internal loop and an additional T.IO split creates an injection volume of 2-3 nl (larger volumes can be injected by on-column focusing with gradient elution separations). The detector uses a U- or Z-shaped flow cell with a 3 nl volume and 8 mm path length. Fused-silica capillary tubing with an internal diameter < 20 pm and zero-dead-volume connectors are used for column connections. (From ref [8j. American Chemical Society).

Figure 10 Enrichment and separation setup for SLM extraction. A - peristaitic pump, B - membrane device with hoiiow fiber instaiied into fused siiica capiiiary, C - iniet on the donor side, D - washing iine used after enrichment, E - microinjection pump to transport the acceptor into ioop F, G - Cis packed capillary column, H - detector window, I - splitter unit, J - high pressure pump. (Adapted from Thordarson E, Palmarsdottir S, Mathiasson L, and Jonsson JA (1996) Sample preparation using a miniaturized supported liquid membrane device connected on-line to packed capillary liquid chromatography. Analytical Chemistry 68 2559-2563.)...

Move the capillary to a horizontal position. Position the filament 1-2 mm below the capillary tip. Move the filament close to the pipet, and heat to bend the capillary by about 15° to the horizontal. One holding pipet lasts one microinjection session and is not reused. Large numbers can be made in advance and stored in a sterile plastic tube. 

Microinjection pipets for physically introducing the DNA fragment into the nucleus of the one-celled egg Microinjection pipets are made from thin-walled glass capillaries with an external diameter of 1mm. Capillaries with an internal filament (Harvard Apparatus Ltd., Edenbridge, UK 30-(X)20) are useful, since they can be backfilled by capillary action from the distal end to the injection tip. Pipets... 

Backfill a freshly drawn microinjection pipet with DNA. Place the end distal to the tip into the DNA solution, allow the liquid to ride into the tip by capillary action and withdraw when liquid can be seen in the tip. Avoid contaminating the DNA stock solution, for example, with glove powder or enzymes from the exposed hand. 

Microinjection pipets are prepared as follows PuU fine glass capillaries with inner filament (Clark Electromedical Instruments, Reading, UK 1.0-mm outside diameter and... 

For microinjection experiments, standard or inverted microscopes are used (e.g., Zeiss Axioskop with transmitted light phase contrast and the mechanical micromanipulator from Leitz (see Fig. 1). The capillary puller is homemade (Fig. 2). The glass tubes (1.4-mm outer and 1.2-mm inner diameters) for preparation of the injection capillaries are obtained from either Schott or Clark Electromedical Instruments. Sterile capillaries can be obtained from Eppendorf. 

Cells are brought into focus and a distinct field is chosen for injection. The capillary is lowered by the manipulator and an individual cell is approached. The cell is injected by further lowering the capillary, and the test material is transferred into the cell by a low pressure exerted with a 50-ml syringe (air-filled) or an automatic injection pump. A small dent is seen at the cell surface when the capillary touches the cell. When the capillary is lowered further, the tip enters into the cell and the dent disappears. Appearance of a white spot indicates that the capillary has penetrated the entire cell and killed the cell. Successful microinjection is marked by slight enlargement of the cell or of the nucleus. 

Adjust the value of the current through the heating filament to your individual needs (usually this value should not vary more than 10% from the value recommended by the supplier) and pull the micropipette. The tip diameter of the micropipette decreases with increasing current and vice versa. Capillaries with an outer tip diameter between 0.2 and 1.0 //m are easily available with the puller used. Two micropipettes will be obtained after 20-30 sec. Both can be used for microinjection because they are identical with respect to their physical parameters. 

Insert the micropipette into the capillary holder of the microinjector (Eppendorf) as for microinjection. 

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... 

Microinjection is a light microscope based technique. The microinjection capillary and its positioning relative to cells are tracked by phase contrast. Moreover, phase contrast is used to identify cell nucleus versus cytoplasm. Microinjection requires in addition to an inverted phase contrast microscope at least two committed pieces of equipment (Fig. 1). These are a micromanipulator to position the injection capillary and a pressure regulator to increase pressure to the capillary selectively as it... 

---