Microinjection is the microscopic-scale manipulation of a living cell or tissue. It is used in a wide variety of research and development applications such as in the cloning of organisms, the study of viruses and cell biology and for treating male subfertility through intracytoplasmic sperm injection (ICSI). The process involves delivering biological materials via a micropipette to target cells or tissues. Microinjection is a form of cellular or pronuclear transfection and can also be used to introduce gene transfer vectors, microRNA and other small molecules.
To perform microinjection, an inverted microscope with a differential interference contrast (DIC) optical system is required. A video camera can be attached to the microscope for documentation of the experiment. Xenopus eggs and embryos are the preferred model organisms for microinjection because of their large size, making them easy to see through an inverted microscope. The embryo is viewed through the microscope at a magnification of 200x and two micromanipulators are used, one to hold the holding pipette and the other to control the injection needle.
The holding pipette is attached to a needle that is lowered into the field of view and directly above the single-cell embryo. The goal is to aim the needle at the pronucleus, a globule of cytoplasm located within the outer layer of the zona pellucida. The needle should penetrate the pronucleus and the cell membrane, without damaging the nucleus. After the needle is positioned, depressing the foot pedal three to four times will inject a red bolus of cytoplasm into the center of the embryo.
After the injection is complete, the micromanipulator controls are used to retract the needle. A 10 microliter pipette tip can then be used to identify the blastomere, a grainy, slightly yellow raised bump of cytoplasm on top of the yolk. It is important to ensure that the blastomere is perpendicular to the needle so the injection has an optimal chance of success.
Once the injection is completed, the embryo is returned to the incubator for overnight incubation. The next day, a microscope can be used to observe the resulting embryos. Healthy embryos will divide to the two-cell stage, and those that are not fertilized or irreversibly damaged by the microinjection will lyse. This will be evident by a swollen embryo and an absence of any movement of the dividing cells.
A good quality microinjection system is essential for maximizing the success of this procedure. Ideally, the injection should be performed using a high-precision microinjection pump that offers both eject and hold pressure. The hold pressure guards against backfilling of the pipette by capillary action and keeps the injectant meniscus close to the needle tip. WPI’s popular PV820 and PV830 Pneumatic PicoPumps are designed for these procedures, with touch screen operation, footswitch operation and a compact design that takes up little bench space.
In addition to microinjection, these systems can be used for other purposes including eukaryotic cell culture and transfection of mammalian cells and rat embryos. This is a great way to study the effect of genes and enhancers on a genome, such as in the emergent model fish species, the threespine stickleback. micro injection