User:Calerc/Pronuclear Injection

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.

Background
Pronuclear injection was developed in the 1980s.

Pronuclear Injection in Mice
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 seperate (i.e., the pronuclear phase). In order to obtain these oocytes, mice are commonly superovulated using gonadotrophins. 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. While efficiency varies, 10-40% of mice born from these implanted oocytes may contain the injected construct. Transgenic mice can then be bred to create transgenic lines.

Problems
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). In addition, if the injected DNA is incorporated in multiple sites, multiple copies of the DNA may be expressed leading to overexpression. 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.