The configuration of any microinjection system will depend upon the application for which it is used. This page lists some of the more common applications for which XenoWorks™ is ideal along with the recommended system configurations.

APPLICATIONS Zygote Pronuclear DNA Microinjection The microinjection of DNA into the pronucleus of a newly-fertilized mammalian egg is now a common and highly efficient method of creating transgenic offspring. Pronuclear microinjection was first described in the mouse, but now many different transgenic animals have been created in this way. Because the micropipette used for injection has an internal diameter typically less than one micron, relatively high pressure (>3000 hPa) is required to inject the DNA solution. Two micromanipulators are required, one to hold the zygote and one to inject the DNA. Gentle negative pressure is used on the holding side, while pulses of high pressure are used to inject 1-2 picoliters of DNA solution into the pronucleus. The XenoWorks Digital Microinjector is ideal for this application, having simultaneous holding and high-pressure injecting capabilities. Suggested system configuration: 1 x Micromanipulator (Right) BRMR 1 x Micromanipulator (Left) BRML 1 x Digital Microinjector BRE110/BRE220 2 x Microscope Adapter Embryonic Stem Cell Transfer into Blastocysts Animals, usually mice, can be engineered with a specific gene function reduced or knocked out altogether by introducing genetically altered embryonic stem cells into the cavity of a blastocyst so that the stem cells contribute to the embryo. The resulting live animal is a chimera of both genotypes. Subsequent selective interbreeding of manipulated animals results in pure-bred gene "knock-outs" or "knock-downs" and can be used for subsequent gene function studies. This operation requires two micromanipulators, one for holding the blastocyst and another for transferring the cells. Both holding and transfer functions require gentle positive and negative pressure for which the Digital Microinjector is ideal. Alternatively, two Analog Microinjectors may be substituted. Suggested system configuration: 1 x Micromanipulator (Right) BRMR 1 x Micromanipulator (Left) BRML 2 x Microscope Adapter 1 x Digital Microinjector BRE110/BRE220 or 2 x Analog Microinjector BRI Intracytoplasmic Sperm Injection Intracytoplasmic sperm injection (ICSI) can be employed for veterinary in-vitro fertilization during rare species preservation or for any veterinary assisted conception. ICSI may also be used as a gene transfer technique when sperm are co-injected with exogenous DNA (see also Piezo-assisted ICSI, below). Typically, two micromanipulators are used, one for oocyte holding and one for sperm aspiration and injection. Each micromanipulator grips a single micropipette holder with a microinjector attached. Since Low positive and negative pressures are required for the delicate task of oocyte holding and sperm injection, an ICSI workstation should be configured with two Analog Microinjectors. Suggested system configuration: 1 x Micromanipulator (Right) BRMR 1 x Micromanipulator (Left) BRML 2 x Analog Microinjector BRI 2 x Microscope Adapter Piezo-assisted ICSI This relatively new technique can be employed for assisted conception in animals in which standard ICSI fails, such as mice. Piezo-assisted microinjection has also been employed as a gene transfer method, where sperm are coated in exogenous DNA and injected into oocytes. The microinjection workstation required for this technique is very similar to standard ICSI, but with the addition of a piezo impact drive attached to the injecting micropipette holder. The device vibrates the injecting micropipette axially and drills its way into the oocyte. This method has been shown to increase success rates. Because the micropipette is vibrating at minute amplitude but high frequency, it is vital to use a mechanically stable micromanipulator, which will not vibrate in sympathy. The more stable the micromanipulator, the more efficient the energy transfer from the piezo impact drive to the micropipette tip. Some piezo-assisted microinjection protocols currently require a bead of mercury inside the injecting micropipette. Please note that mercury should not be used in conjunction with the Digital Microinjector, though the Analog Microinjector is ideal for this purpose. Suggested system configuration: 1 x Micromanipulator (Right) BRMR 1 x Micromanipulator (Left) BRML 2 x Analog Microinjector BRI 2 x Microscope Adapter 1 x PMM-150FU Microinjection of Cultured, Adherent Cells Cultured cell lines such as 3T3, CHO and HeLa can be microinjected while attached to a Petri dish. The procedure is best viewed through phase-contrast optics; a single micromanipulator and a single high-pressure microinjection channel are required. The tip of a sharp (inner diameter less than 1 micron) micropipette is brought down on top of a single cell and a pulse of high (100-1000 hPa) pressure applied. The cell membrane is ruptured and the cell can be seen to inflate slightly. Volumes injected are typically less than 5% of the cell volume. Success rates vary widely depending upon the type and volume of compound injected, the culture conditions and the cell line used. The high-pressure functions of the Digital Microinjector and the smooth, fine control of the micromanipulator are particularly well-suited to this application. Suggested system configuration: 1 x Micromanipulator (Right) BRMR 1 x Digital Microinjector BRE110/BRE220 1 x Microscope Adapter Somatic Cell Nuclear Transfer The enucleation of an oocyte followed by the transplantation of a somatic cell is a method of producing genetically identical copies (clones) of the animal from which the donor cell was taken. Generally, two micromanipulators are required, one for holding the oocyte and another for the enucleating and injecting procedures. Eash micromanipulator grips a single micropipette holder with a microinjector attached. Oocyte holding, enucleation and somatic cell transplantation require gentle, low positive and negative pressure, making the Analog Microinjector ideal. Suggested system configuration: 1 x Micromanipulator (Right) BRMR 1 x Micromanipulator (Left) BRML 2 x Analog Microinjector BRI 2 x Microscope Adapter