Grafting pipets

Injection pipets with an inner diameter of about 1-2 im are made from thin-walled glass capillaries. The internal diameter of grafting pipets is larger and varies according to the size of the clumps of cells to be grafted. 

Beveled pipets are made from the grafting pipet made in step 3 (Fig. 3D Notes 10 and 11). Touch one side of the pipet with the heated glass bead. When the pipet tip fuses with the bead, slowly withdraw the pipet to puU a sharp bevel of about 5 pm in length. 

Pick up the cell clumps into the grafting pipet with a small amount of medium. Draw the cell clumps about 20 jm into the grafting pipet. 

Insert the injection and grafting pipet through the yolk sac then the amnion into the amniotic cavity. 

If the internal diameter of the grafting pipet is large relative to the size of the embryo, it may be difficult to puncture the tissue layers with the pipet during grafting. Beveled pipets can be used in this situation. The sharp point of the beveled pipet slices the tissue layers to create a passage for the grafting pipet. 

Excel macro generating pipetting tables from a list of sequences is available online (www.5z.com/mlebl/macros.html). The area of grafted solid supports in peptide synthesis was recently reviewed (17). 

Fig. 1. Tools used for micromanipulation of 6.5-8.5-d mouse embryos. A The glass needle, B the holding pipet, C the injection pipet used for delivering dye or DNA solution, D the pipet used for grafting cell clumps, and E the two bends produced in the pipet (shown in B-D) to enable the tip of the pipet to reach over the wall and access the medium drops sitting on the bottom of the culture dish during micromanipulation (for setting up, see Fig. 5B).

Fig. 9. Grafting of cells to a 6.5-d embryo. The embryo is held on the anterior side opposite the intended site of grafting by the holding pipet (h). The cells are grafted into the epiblast (ep) on the posterior side of the embryo ( marks the position of the primitive streak) immediately proximal to the margin of the distal cap. Bar = 20 pm.

Apply a positive pressure to the de Fonbrune syringe to expel the graft from the pipet as the pipet is slowly withdrawn from the anbryo (Notes 25-27). A coordinated movement of the pipet and the expulsion of the graft is critical to the precise positioning of the graft in the epiblast. 

Angled pipets are used for holding and grafting cells into the embryos. Place the pipets in the instrument holders of the manipulator. Tilt the manipulators so that the tips of the pipets are immersed in the paraffin oil and point to the bottom of the petri dish. Figure 6 shows the setup for manipulating 7.5-d embryos. 

Fig. 3. Manipulations under the microscope. A Host embryo (stage 3+). B-D Clear yolky endoderm to expose the epiblast at the future grafting site. E Donor embryo, ventral side uppermost (stage 4). F-J Cut out Hensen s node from the donor. K and L Pick up the excised node with a Gilson pipet. M-N Manipulate the node to the graft site. O The finished operation.

When the graft is in position (approximately) (F. 2L), very carefully remove most of the fluid from above the embryo with a Pasteur pipet while watching under low power (Fig. 3A). If necessary, reposition the graft with a fine mounted needle (but this is quite difficult if there is no liquid above, as the graft tends to stick to the needle). 

Add about 100 pL of antibiotic-antimycotic concentrate (away from the graft site) with a Gilson (Fig. 3D) or Pasteur pipet (1-2 drops). 

Use the tungsten needle to gently separate the neural plate from the paraxial mesoderm Roll off or push away the neural plate as it comes loose (Note 6). Rinse the operated area with saline-PS from the 1-mL syringe to wash off the Dispase. The aim of this procedure is to make a pocket beneath the lateral part of the neural plate to house the notochord implant in close apposition with the basal surface of the neuroepithelium. Therefore, do not fully separate the neural plate and the paraxial mesoderm, as the graft will then settle down too deeply next to the host notochord. Cut donor notochords into pieces of about 5(X)-l,000 jm, transfer one piece in as little volume as possible to a serum-coated 20-pL pipet tip. 

Retrieve the neural tissue piece to be grafted from the petri dish using a P20 pipet-man and yellow tip set on 10-20 pL. If there are problems with the tissue getting stuck to the plastic pipet, you can coat the inside of the yellow tip with sterile serum before use. 

Transfer polarizing tissue graft into host egg using either a glass or gilson pipet or a very small spatula. 

---