For this reason, mice, rats or other small mammals are used to introduce foreign DNA into oocytes or embryos (blastocysts).Two different techniques are used: Microinjection of linear DNA into the pronucleus (Fig. 1) Transfer of Embryonic Stem Cells into Blastocysts (Fig. 2)
(one cell embryo, pronuclear stage)
Microinject gene of interest as linear DNA into male nucleus
Introduce several such embryos into pseudopregnant mouse
ES cells injected into cavity of blastocysts
Injected cells become incorporate d in inner cell mass of host blastocyst
Introduce several such blastocysts into pseudopregnant mouse
If this does not occur, only a few cells will integrate the gene. For this reason, the DNA is introduced into the fertilized egg at the earliest stage, which is the pronuclear period immediately following fertilization. For several hours following the entry of the sperm into the oo cyte, the male and the female pronuclei are visible as individuals under normal light microscope and not fused into a so-called zygote.
The DNA may be injected into either of these pronuclei with no difference in results. Usually the injection is into the male pronucleus because it is larger than the female nucleus and also because it is closer to the oocyte surface.
These oocytes are subsequently transferred into the uterus of pseudopregnant
recipient animals and develop to term.
The actual holding of the embryo is carried out using CellTram Air.
Two possibilities are available for injecting DNA:
using the CellTram Air as a simple injection device or using the FemtoJet for reproducible and programmable injections.
Together with an inverted microscope, combined with Hoffmann modulation
contrast optics or
differential interference contrast optics, microinjection can be effected quickly and simply.
The capillary can be moved easily in any direction (x y z) by means of a joystick. By simply pressing the joystick button, the capillary can be moved repeatedly to one of the two pre-programmed positions.
The setting of these positions depends on how the TransferMan is used (Fig. 4)Fig. 4 TransferMan for Transgenic Animals DNA-Injection Left-hand side - holding function: The area where the uninjected embryos are placed is set to Position 1. Here one embryo can be taken easily by using CellTram Air and VacuTip. This embryo is moved via the joystick to a central position in order to inject the DNA.
Following DNA injection, the capillary is moved to a parking position (can be outside the view). This position is defined as Position 2. Pressing the joystick button moves the capillary back to Position 1. The procedure then restarts.Right-hand side - injection position: The area where the injection capillary is kept for injection is defined as Position 1. Position 2 is its Home position.
The capillary holder is attached either to CellTram Air or to the
Femtotips are available in two shapes for injecting cultured cells or suspended cells. Backfilling with an Eppendorf Microloader easily places the injection material directly into the end of the Femtotip.Transfer of Embryonic Stem Cells - Injection of ES cells into the cavity of blastocysts using a microscope (inverted), micromanipulator equipment and transfer / holding devices .
Embryonic stem cells are derived from the inner cell masses of normal blastocysts (early embryos, mostly mouse embryos). These cells are pluripotent, which means they can develop into any type of tissue. Therefore, removing ES cells from the culture and placing them back into an early embryo (blastocyst) allows them to divide and become part of the embryo. Transfection allows foreign genetic material to be inserted in vitro into these cells. The target of this process is homologous recombination with the chromosome of the cell, i.e. introduction or exchange of DNA at one location with homologous DNA sequences. Following transfection, the descendants of one individual cell (clone) are analyzed using microbiological methods to ascertain whether any mutation is present. Cells of the identified clones which showed the desired reaction are multiplied in vitro and injected into blastocysts.
These injected blastocysts are then implanted into the uterus of pseudo-pregnant females.Setup of the workstation: As for the injection of DNA, two TransferMan devices are used for controlling the holding and transfer capillaries. The actu al holding of the blastocysts is performed by using CellTram Air. CellTram Oil is used for transferring the ES cells. As aforementioned, one inverted microscope, in combination with Hoffmann modulation contrast optics or differential interference contrast optics, makes the ES cell transfer quick and simple. The setting of the positions is carried out as shown in Fig. 6:
After ES cells have been injected, the capillary is moved to a parking position (can be outside the view). This position is defined as Position 2. Pressing the joystick button moves the capillary back to Position 1. The procedure then restarts.Preparation of the petri dish: Blastocysts are placed onto a petri dish in one or more drops of medium. Blastocysts should be positioned close together in one area of the drop. The ES cells are placed in another drop. All drops on the dish are covered with oil. The method of procedure is as follows: The transfer capillary is directed to the focus level and the position is stored as Position 1. Position 2 is defined beside or above this drop.
The drop with the ES cells is placed in focus and several ES cells are aspirated into the transfer capillary by rotating the knob of the CellTram Oil. The transfer capillary, which now contains the cells, is moved up into the overlay medium. The petri dish is moved in order to bring a blastocyst in the other drop into view. One blastocyst is taken with the second TransferMan (the holding capillary) in the Position 1 and moved to the injection position. In this position, the ES cells are injected into the cavity of this blastocyst.
The injected blastocyst is stored in Position 2 by simply pressing the joystick button of the manipulator on the left-hand side.
Both techniques can be easily performed with the Eppendorf Micromanipulator TransferMan, CellTram Air, CellTram Oil and the FemtoJet.
The automation of repetitive tasks while maintaining easy operation allows the user to concentrate on the micromanipulations themselves. This enables rapid work with good results. Replace one TransferMan with an InjectMan and expand the versatility of the system to include adherent cell injection.Literature  Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A., Ruddle, F.H.: Genetic transformation of mouse embryo by microinjection of purified DNA. (1980)Proc.Natl.Acad.Sci USA 77, 7380-7384