A micro injection is the use of a glass micropipette to inject liquid substance at a microscopic or borderline macroscopic level (this includes living cells and intercellular space). Microinjections are typically performed under an inverted microscope with magnification power of 200x or less, but can also be done under a dissecting stereo or traditional compound microscope at similar magnifications. Microinjections can be used to inject a variety of different substances including chemicals, dyes, enzymes, viruses and RNA.
The most common application for microinjection is the generation of transgenic mice. This is achieved by injecting a DNA construct into the male pronucleus of fertilized mouse embryos to replace its natural gene with one that encodes a fluorescent protein. The injected DNA is then expressed as a chromophore in the outer zona pellucid layer and cell membrane of the embryo.
Embryos that successfully divide to the two-cell stage following overnight incubation are then transferred into the oviduct of a pseudopregnant recipient female. Most injected embryos will develop correctly, but some may arrest at the one-cell stage or show a fragmented appearance due to incorrect nuclear migration (Fig. 8.8A). These embryos are lysed, leaving behind a subset of correct dividing embryos that can be sorted by PCR for the presence of the desired gene.
The success of this method of gene transfer depends on a number of factors including the size of the DNA construct, the ability to target the male pronucleus, and the ability to avoid the formation of mosaic founders. In order to improve the genetic transformation efficiency of mouse embryos, Creative Biolabs has developed a new technique called pinpoint cell penetration microinjection. This technique is more accurate and precise than conventional electroporation, allowing for greater gene delivery per cell.
In order to perform a microinjection, it is important that the experimental environment is well-controlled. In most cases, the experiment will be carried out using a microscope equipped with a micromanipulator that controls the needle that is used to insert the injection. A specialized software is used to control the needle movement and provide feedback that helps to ensure that it is positioned at the proper focal point of the embryo.
There are a number of techniques that can be used to perform a microinjection, including valves, moving injectors and rotating injectors. These methods are expensive, time-consuming and require experienced operators to be effective. They also have low productivity and success rates compared to passive microinjection. Passive microinjection, however, is a simple, cost-effective and reliable method for gene transfer to cells and organisms. micro injection