Microinjection micropipettes

Nuclear transplantation technique. The nucleus of an unfertilized egg is mechanically removed with a micropipette (.left), and a nucleus from a blastula cell is removed and microinjected into the enucleated egg. 

The majority of the transgenic mice produced to date have been through the microinjection route, which was used for the production of the first transgenic mouse (Isola and Gordon, 1991). In this procedure, fine glass micropipettes are used to introduce extremely small volumes of DNA solutions into the pronucleus of... 

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. 

The protocol described below allows characterization of the tip diameter by bubble pressure measurement (Mittman et al., 1987). This is a nondestructive method for determining tip size, and micropipettes can still be used for microinjection experiments without any restrictions. This is not the case when micropipettes are characterized by electron microscopy. 

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

Increase the pressure of the microinjector until air bubbles from the micropipette tip appear, indicating that the tip is open. Typical pressure values for good micropipettes, used in our laboratory for nuclear and cytoplasmic injections, are between 2000 and 4000 hPa. Lower values indicate a tip size above 1 //m. For higher pressure values the micropipette is either closed or the risk for clogging during microinjection is rather high. 

To avoid micropipette clogging during microinjection, always centrifuge the sample for at least 10 min at 10,000 g at 4°C. 

The microinjected volume varies with the pressure applied to the micropipette and the time (controlled by computer) the tip stays inside the cell. Further, the volume depends on the cell type and injection solution used. Typical values are 0.1-0.5 pi for cytoplasmic and 0.01-0.05 pi for nuclear injections. 

Celis, J. E. (1984) Microinjection of somatic cells with micropipettes Comparison with other transfer techniques. Biochem. ]. 223, 281-291. 

Substratum attached cells. My experience is entirely with either substratum attached HeLa or Vero cells. The more spread the cell the better. Depending on purpose, the cells may be grown either on tissue plastic, glass coverglass onto which cells are seeded in a tissue culture dish, or cover glass bottomed tissue culture dishes (MatTek). Cells should ideally be 50-70% confluent for short-term, 6-10 h post injection experiments. Microinjection can be done with suspended cells. Successful suspended cell injection requires a two micropipette-system in which one pipette is used to hold the cell and another to inject the cell. 

Fig. 3. Steps in capillary micropipette manipulation. A. Backloading of capillary micropipette with solution (arrow points to Geloader tip inserted into back opening of micropipette). B. Capillary micropipette in place over open cell culture media (arrow points to capillary micropipette). C. Low power appearance of capillary micropipette in culture media (arrow points towards slightly out of focus capillary tip and phase bright spot is where capillary enters the culture media). lOx objective, phase contrast. D. Appearance of capillary micropipette as it enters the cytoplasm of a HeLa cell. Cytoplasmic microinjections are near the nucleus as the ceU is thickest in this area and hence the target depth is greatest.

A detailed discussion of suitable microinjection equipment and technical aspects is beyond the scope of this chapter and are described in ref. 3. The glass micropipettes used for penetration of cells and delivery of the sample are avail-... 

Figure 18.1. Micromanipulation slide designed for upright microscopes. (A) A perspective drawing of the metal microinjection support slide (for a mechanical drawing, see Kiehart 1982). (S) The assembled injection chamber showing top and bottom coverslips in place, the microscope stage, and part of a micropipette. The arrow points to the position of the embryos near the front edge of the bottom side of the top coverslip.

Align the micropipette perpendicular to the region of the embryo to be microinject-ed. 

Fig. 1 Microinjection of zebrafish embryos, (a) A typical setup for microinjection. (b) Recently fertilized eggs. The insets show early left) and late right) phases of one-cell-stage embryos, (c) Microinjection plate an acrylic plate with V-shaped grooves for holding embryos, (d) One-cell-stage embryos are arrayed in the grooves for microinjection. Scale bar, 2 mm. (e) DNA constmcts are injected into an embryo by penetrating the blastomere with the micropipette through the chorion. Scale bar, 200 pm...

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