Cre-lox Recombination: A Tool for Cell-Specific DNA Manipulation

Cre recombinase is an enzyme derived from the P1 bacteriophage that recognizes a specific type of DNA segment called a LoxP site. Cre recombinase will manipulate DNA that is located between two LoxP sites depending on the LoxP orientation.

EXCISION

INVERSION

TRANSLOCATION

Cre recombinase performing excision, inversion, and translocation

Excision

Cre recombinase will cut out or “excise” DNA between two LoxP sites that run in the same direction. This method can be used to create a knockout mouse model that will be missing the gene of interest. Researchers can then see what happens when the mouse no longer has the gene to infer its function. This method is advantageous when studying a gene whose absence can be lethal to the prenatal development of a mouse but not lethal once the mouse has developed.

Same-orientation LoxP sites can also be used to activate gene expression. A STOP codon that is placed upstream of a gene will prevent that gene from being transcribed by RNA polymerase. Cre-recombinase can excise the STOP codon if it is surrounded or “flanked” by LoxP sites of the same orientation, allowing the downstream gene to be expressed. 

Inversion

Cre-recombinase will invert DNA that is between two LoxP sites of opposite orientation. This method is often used with adeno-associated viruses containing a gene that researchers want expressed in specific cell types. The idea is to inject an adeno-associated virus that contains an already inverted (inactive) gene flanked by two LoxP sites of opposite orientation  into the brain region of interest. Cre-recombinase will recognize the LoxP sites and invert the gene, subsequently flipping it to an active, readable form for transcription. Neuroscience researchers often use transgenic mice expressing Cre-recombinase in a specific cell type to limit Cre-recombinase activity to the cell type they are interested in studying. This is usually done by tagging Cre-recombinase with a cell type-specific promoter (e.g. drd1-Cre for dopaminergic neurons). This approach allows temporal control of gene expression in a specific area.

Translocation

LoxP sites located on different chromosomes result in the translocation of genetic material of homologous or non-homologous chromosomes. Chromosomal translocations can cause cancer, so perhaps Cre-recombinase can be used to study these translocations or how to “fix” chromosomal translocations that have already occurred.

Cre-lox recombination is essentially a tool to manipulate endogenous DNA expression and to introduce exogenous genes to better understand neural systems and molecular pathways, and thus has become a very popular method in studying transgenes and gene function. However, Cre-recombinase is much more complex than this article aims to explain. For those who want to learn more, I suggest checking out this link or doing more research yourself!