Cre-loxP Recombination Essentials Part 1

You might have heard of the Cre-loxP system even if you are not directly working with genetic manipulation. The Cre-loxP system is an ubiquitous technology for genetic manipulation and a mainstay in mouse research labs. With this system you can delete genes in cells, specific tissues and even whole organisms! You can start to master this system by understanding its two main components:

The Cre or cre-recombinase protein (called so because it causes recombination) catalyzes site-specific recombination events between two DNA recognition sites. This recombinase comes from the P1 bacteriophage.

The Lox or loxP sites (locus of X over P1) are the palindromic sites recognized by cre-recombinase. Each site is 13 bp long, and an 8 bp spacer region separates the two sites. DNA sequences found between the two loxP sites are said to be floxed (or flanked by loxP).

Depending on the position and orientation of the loxP sites, three recombination events may occur:

1. Inversion. This occurs when the two loxP sites are on the same chromosome and in opposite orientations i.e. the floxed DNA sequence is inverted in reverse order.
2. Deletion. When the two loxP sites are on the same chromosome and in the same orientation the floxed DNA sequence will be deleted.
3. Translocation. If the two loxP sites are on different chromosomes and in the same orientation a translocation event will cause exchange of DNA segments.

Even though researchers use all 3 of these events, deletion is usually the event of choice in mouse genetics. Some of its most popular applications include:

Global Gene Deletion in Every Cell in the Mouse

To avoid the tedious job of creating a complete knockout mouse you can make a global Cre-loxP gene deletion system.

• Here, you would have loxP sites flanking your gene of interest (let’s call it loxP-GENE-loxP) in one mouse strain and the cre-recombinase produced under a global promoter (present in all cells, e.g. EIIa or Sox2) in another mouse strain.
• Crossing the two strains to obtain a homozygous cre-flox strain will result in global deletion of your gene of interest.

How is this an advantage over generating a complete gene knockout mouse, you ask? Well, you can use the flox mouse (the line not crossed to the cre line) for other purposes too (see below).

Cell-, Tissue- or Organ-Specific Gene Deletion in the Mouse

To examine how your gene functions in a particular cell or tissue type or organ, you can take the loxP-GENE-loxP mouse described above and cross it to a mouse strain which expresses cre-recombinase under a cell-, tissue- or organ-specific promoter. This will delete your gene of interest only in the specific system under study, thus giving a more focused answer to your research questions.

As an example, you would use a CD4-cre mouse to study helper T-cells or an Alb-cre mouse to study deletion in the liver, thus effectively eliminating effects seen due to deletion in other cell systems not under consideration.

Delete a Gene at a Particular Time Point With an Induction Stimulus

Sometimes a complete gene knockout mouse could be lethal if the gene deleted is essential for embryonic development or other crucial cell functions. In such cases, an induced Cre-loxP system is ideal.

• The cre-recombinase is again under the control of a required promoter (global or cell-specific), but is also attached to a mutated ligand-binding domain of the estrogen receptor that prevents the cre protein from translocating to the nucleus.
• In the cre-ER-loxP-GENE-loxP mouse you can now induce deletion at any time point you want by simply treating your mice with Tamoxifen (a synthetic estrogen analog). It binds to the mutated estrogen receptor causing it to translocate to the nucleus, thus helping the cre protein to recombine DNA between loxP sites.
• Another variant to the Tamoxifen system is the Tet-ON system that uses Doxycycline (a Tetracycline analog) to induce cre expression.

The inducible system is an invaluable way to study gene deletions at particular stages of development or the cell cycle.

Study Gain-Of-Function Mutations

Another interesting use of the Cre-loxP system is studying gain-of-function instead of loss-of-function as described in the previous examples. Imagine that you place a STOP codon before a gene of interest thus preventing its transcription and translation in cells. Go ahead and flank the STOP codon with loxP sites. Now if you cross such a loxP-STOP-loxP-GENE mouse to your desired cre mouse strain, the recombination event would lead to deletion of the STOP codon and thus expression of the gene in only the required cell types (where you expressed your cre protein). In this way, you can study the activity of your gene of interest in a specific cell type.

Track Your Cells

Throw in a fluorescent protein after your gene of interest and watch the turning on or off of the gene with cre recombination. A fascinating use (with some clever modifications) of this technique is the Brainbow or Confetti mouse. Labs that design cre mice under various promoters use loxP-GFP-loxP or another flanked marker protein to test efficiency of the promoter that is driving the cre protein. In this way, they can assess the specificity of cre activity.

After reading about the wonderful applications of this technology, I am sure you will want to try your hand at it. Stay tuned for a follow up article about the common pitfalls of this system and ways to get around them.

Do you use Cre-loxP in your research? Do you know some other applications than those mentioned here? Please share your experiences with us by writing in the comments section!