Microinjection
Microinjection refers to the process of using a glass micropipette to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the nuclear envelope. The desired contents are then injected into the desired sub-cellular compartment and the needle is removed. Microinjection is normally performed under a specialized optical microscope setup called a micromanipulator. The process is frequently used as a vector in genetic engineering and transgenics to insert genetic material into a single cell. Microinjection can also be used in the cloning of organisms, and in the study of cell biology and viruses.
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Subtypes [edit]
Pronuclear Injection [edit]
Pronuclear injection is a technique used to create transgenic organisms by injecting genetic material into nucleus of a fertilized oocyte. This technique is commonly used to study the role of genes using mouse models.
Pronuclear Injection in Mice [edit]
In order for pronuclear injection to be successful, the genetic material (typically linear DNA) must be injected while the genetic material from the oocyte and sperm are separate (i.e., the pronuclear phase).[1] In order to obtain these oocytes, mice are commonly superovulated using gonadotrophins.[2] Once plugging has occurred, oocytes are harvested from the mouse and injected with the genetic material. The oocyte is then implanted in the oviduct of a pseudopregnant animal.[1] While efficiency varies, 10-40% of mice born from these implanted oocytes may contain the injected construct.[2] Transgenic mice can then be bred to create transgenic lines.
Problems with Pronuclear Injection [edit]
The incorporation of injected DNA into the mouse genome cannot be tightly controlled. As a result, improper incorporation may occur. If DNA is incorporated at the 2-cell stage, some cells may express the DNA while others will not (so called mosaic mice).[2] In addition, if the injected DNA is incorporated in multiple sites, multiple copies of the DNA may be expressed leading to overexpression.[2] Even if only one copy of DNA is incorporated into the genome, if it is not incorporated into the germline, then it will not be passed to offspring.[2]
Protoplast Injection [edit]
Microcapillary and microscopic devices can be used to deliver DNA into a protoplast.[3]
References [edit]
- ^ a b Mullin, Ann. "Pronuclear Injection". Tulane University.
- ^ a b c d e "Pronuclear Injection". UC San Diego. Retrieved 21 February 2013.
- ^ http://opbs.okstate.edu/~melcher/MG/MGW4/MG433.html
See Also [edit]
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