A transgenic animal is a artificial mutant caused by the introduction of a foreign DNA sequence leading to a genetic change in the genome (ATBW Empfehlung, 1994).
Genetically modified DNA sequences can be incorporated at random into the genome (via pronuclear injection), or incorporated at a specific location (via ES cell targeting and blastocyst injection). The exogenous DNA can express a functional protein or change the expression of an endogenous gene (e.g. Knock-out).
Mainly 2 methods are used to generate genetically modified mice.
Pronuclear Microinjection into Zygotes
The microinjection of DNA directly into the pronuclei of fertilized zygotes is a common method to transfer genes into the mouse. After injection the embryos will be transferred into the oviduct of a foster mouse. Part of the embryos will get born. Some of the newborn have integrated the exogenous DNA into the genome and are therefore called transgenic.
Microinjection of ES cells into Blastocysts
ES cell gene targeting
Embryonic Stem cells are cells that (originally) have been isolated from the inner cell mass of a mouse embryo. In an undifferentiated state these cells can be cultured indefinitely. During ES cell culture / targeting the cells are grown on feeder cells (inactivated mouse embryonic fibroblasts (MEF)) in the presence of LIF (leukaemia inhibitory factor). In this way the ES cells do not differentiate and can be electroporated with a DNA targeting construct in order to genetically change a gene of interest.
Microinjection into blastocysts
The electroporated ES cells are injected into blastocysts, whereby the cells integrate into the developing embryo. The advantage of this technique is that one can select the correctly targeted ES cells before injection. With this method it is possible to generate mice with mutations in specific genes (e.g. Knock-out mice).