Microinjection Applications

1. Zygote Pronuclear DNA Microinjection
The microinjection of DNA into the pronucleus of a fertilized mammalian egg has become a frequently used approach to produce transgenic offspring. Although pronuclear microinjection was initially carried out in the mouse, several different transgenic animals have now been made using this method. As the micropipette used for injection is sharp, relatively high pressure (> 3000 hPa) is needed to transfer the DNA solution.

This application requires the use of two micromanipulators; one to hold the zygote and the other to inject the DNA. A subtle negative pressure is applied on the holding side, whilst rhythms of high pressure enable injection of the DNA solution into the pronucleus. Due to its concurrent holding and high-pressure injecting abilities, the XenoWorks digital microinjector is perfect for this application.

2. Embryonic stem cell transfer into blastocyst
Introducing genetically altered embryonic stem cells into the cavity of a blastocyst causes the stem cells to transfer genetic material to the embryo. The resulting animal is engineered with a combination of both genotypes. Two micromanipulators are needed for this process; one for holding the blastocyst and one for moving the cells. Both functions require mild positive and negative pressure. Although the digital microinjector is ideal for this, two analog microinjectors can be used as an alternative option.

3. Somatic Cell Nuclear Transfer
Genetically identical copies of an animal can be achieved with enucleation of an oocyte followed by the transfer of a somatic cell. Two micromanipulators are typically needed for this; one for holding the oocyte and the other for the enucleating and injecting processes. Each micromanipulator attaches to a solitary micropipette holder with a microinjector connected. Mild positive and negative pressure are required for oocyte holding, enucleation and somatic cell transplantation, of which analog microinjectors are therefore appropriate.

4. Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection (ICSI) is a vital process used for veterinary fertilization in-vitro, which can aid the conservation of rare or endangered species. Less commonly, ICSI is used as a gene transfer procedure whereby sperm are co-injected with exogenous DNA.

Two micromanipulators are used for this; one for oocyte holding and the other for sperm aspiration and injection. As mild positive and negative pressures are needed for the intricate jobs of oocyte holding and sperm injection, an ICSI workstation ought to be configured with two analog microinjectors.

5. Piezo-assisted ICSI T
This is new approach is developed to aid conception in animals when standard ICSI has previously failed. It can also be used for gene transfer, whereby sperm are covered in exogenous DNA and subsequently injected into oocytes. The microinjection workstation required for this is similar to that used for standard ICSI but necessitates a piezo impact drive connected to the injecting micropipette holder.

In this approach, the micropipette vibrates and drills into the oocyte. The small amplitude yet high frequency of this vibration means a mechanically stable micromanipulator must be used. Good stability will ensure efficient energy transfer from the piezo impact drive to the micropipette tip.

Most piezo-assisted microinjection protocols require a bead of mercury to stabilize the injecting micropipette – mercury should not be used in combination with a digital microinjector, but the analog microinjector, on the other hand, is suitable.

6. Microinjection of Cultured, Adherent Cells
Cultured cell lines can be microinjected whilst still attached to a Petri dish. The best way to view this procedure is using phase-contrast optics with a micromanipulator and a high-pressure microinjection channel. This process involved bringing down the tip of a sharp micropipette onto a single cell before a pulse of high pressure is generated. The high-pressure ability of the digital microinjector along with the smooth control of the XenoWorks micromanipulator are both ideal for this application.