Microinjection is the injection of a liquid element at a microscopic scale into a target cell or intercellular space. DNA, RNA and other biological molecules are most commonly injected into cells using this technique for gene transfer and expression. Although a number of chemical and physical methods for introducing genetic material into cells have been developed over the years, microinjection is currently the most direct approach.
In microinjection, the genetic material is introduced into a live cell using very small bore glass needles with an outer diameter of less than 0.2 mm. To perform the procedure, the needle is lowered into a field of view under an inverted microscope and positioned over the oocyte or embryo. The needle is then manipulated to pierce the oocyte or embryo with a very thin needle tip (typically less than 1 m in diameter). After the oocyte or embryo is punctured, the needle is filled with an aliquot of the DNA and inserted into the cell to introduce the genetic material.
A large number of different DNA insertion methods exist, but microinjection is the most direct method and allows for accurate control over the number of inserted genes per cell. In addition to DNA insertion, other material can also be introduced into the cell, including proteins and lipids.
The process of microinjection is a critical step in the creation of transgenic organisms. Inserting new genetic material into a fertilized oocyte transforms the resulting offspring to possess characteristics of a different species, a phenomenon known as horizontal gene transfer. This can occur between two different species or between the same species and a wild animal, resulting in a chimera.
Unlike other methods for delivering genetic material into cells, such as electroporation, which opens many pores in the cell membrane, microinjection results in precise puncture of the cell membrane and significantly increases survivability of the embryo after the procedure. This is because the pinpoint cell penetrator focuses on only one point of the oocyte or embryo, instead of opening up multiple pores as happens with electroporation.
Microinjection allows for more precise delivery of the DNA or other material to the cell, and can be done at a much lower temperature than most electroporation techniques. This is a crucial factor in human embryos, which are extremely sensitive to high temperatures and can be irreversibly damaged by electroporation.
To achieve this, the needle is lowered into a live embryo at a low, controlled velocity. Several steps are taken to optimize the injection conditions for the best results. First, the oocyte or embryo must be selected for the correct stage of development. This can be accomplished by using ‘Assisted Hatching’ techniques, which help to select the highest quality eggs or sperm for microinjection.
The next step is to lower the needle at a slow and constant pressure, so as not to damage or disturb the embryo. Ideally, the needle should pierce the corion and be positioned directly over the blastomere, which is a grainy, slightly yellow bump of cytoplasm on the top of the oocyte yolk. Once the needle is lowered, it should be depressed several times to inject so that a red bolus of DNA with diffused edges fills about 1/8 of the embryo’s cytoplasm. micro injection