Introduction

Definition

Genetically engineered (transgenic) mice are mice with an altered genome through the use of genetic engineering techniques.
 
Genetically modified DNA sequences can be incorporated at random into the genome (via pronuclear injection), or incorporated at a specific location (CRISPR/Cas or 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-in, Knock-out).

Mainly 3 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.

CRISPR/Cas (endonuclease mediated genome editing)

CRISPR/Cas (endonuclease mediated genome editing): RNA (gRNA and Cas9 mRNA) and DNA (Template) are injected into the pronucleus or the cytoplasm of fertilized mouse egg cells (zygotes). After injection the embryos will be transferred into the oviduct of a foster mouse. Part of the embryos will get born. After a process called non-homologous end joining (NHEJ) some of the newborn mice could have an indel (insertion or deletion of a few nucleotides) at a specific location. After a process called homology directed repair (HDR) some of the newborn might have integrated a DNA sequence (Stop codon, loxP, GFP etc.) at a specific location in the mouse genome.

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).

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