You might have heard of the Cre-loxP system even if you are not directly working with genetic manipulation. The Cre-loxP system is a ubiquitous technology for genetic manipulation and a mainstay in mouse research labs, mainly used for the generation of mouse knockouts.
However, as with any other technology or research tool, it has limitations and pitfalls that need to be considered while planning experiments or interpreting results.
This article will introduce the concept and applications of Cre-loxP, take you through some of the technology’s pitfalls, and provide potential solutions to solve your Cre-loxP challenges.
For a recombination-based approach to markerless gene deletion in bacteria, see old reliable: two-step allelic exchange instead.
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The Two Main Components of the Cre-LoxP System
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).
Three Possible Recombination Events
Depending on the position and orientation of the loxP sites, three recombination events may occur:
- 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.
- Deletion. When the two loxP sites are on the same chromosome and in the same orientation the floxed DNA sequence will be deleted.
- 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.
Five Applications of the Cre-LoxP System in Mouse Genetics
Some of the most popular applications of this system include:
- Global Gene Deletion in Every Cell in the Mouse
- Cell-, Tissue- or Organ-Specific Gene Deletion in the Mouse
- Delete a Gene at a Particular Time Point With an Induction Stimulus
- Study Gain-Of-Function Mutations
- Track Your Cells
1. 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).
2. 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.
3. 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.
4. 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.
5. Track Your Cells
Throw in a fluorescent protein after your gene of interest and watch the gene turn on or off with Cre recombination. A fascinating use of this technique (with some clever modifications) 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 the efficiency of the promoter that is driving the Cre protein. In this way, they can assess the specificity of cre activity.
Common Pitfalls With the Cre-loxP System:
| Pitfall | Description |
|---|---|
| Cre Toxicity | We often hear that too much of anything is bad, and this is true for the Cre protein. Several researchers have found that excessive cellular accumulation of Cre recombinase can lead to DNA damage and cell death.2 Affected mouse strains exhibit decreased viability and infertility. This pitfall is a serious problem in certain cell types (Cag-cre, CD2-cre) but not in others. |
| Cre Mis-Recombination | Cre recombinase may target sites in the genome that are similar to loxP sites (cryptic loxP sites), thus inducing recombination or deletion events at non-specific sites. This could lead to disruption of important or essential genes and, thus, causing cell or organism death or, at the very least, unexpected cellular phenotypes. Always use the Cre mouse as a control to help distinguish off-target effects. |
| Cre Non-Specificity | Even if your mouse strain expresses Cre under a specific promoter, the Cre promoter might be leaky and express Cre in cells that don’t contain that promoter. Such non-specific Cre expression can lead to confounding results with regards to cell-specific gene targeting. |
| Cre Mosaicism | Variable or inconsistent Cre expression has been observed in different cells or tissues of the same mouse leading to inefficient deletion of floxed genes (e.g., Vav1-re or Fabp4-cre).3 This causes a phenomenon known as Cre mosaicism. This could lead to problems where littermates in a group that should show same results in an experimental setup will show inconsistent observations due to inherent changes in their Cre-related phenotypes. |
| Parent-Dependent Cre Expression | Cre activity depends on whether Cre comes from the male or the female parent. In some strains (e.g., EIIa-cre)3, Cre is more efficient in deletion when inherited from the maternal side. To track the Cre inheritance pattern of your Cre mouse strain, read the available reports/publications about your strain. Otherwise, compare the Cre efficiency using mice offspring obtained from a Cre mother only and a Cre father only. If considerable differences are seen between the two kinds of offsprings, use the one with the superior Cre efficiency. |
Circumventing the Pitfalls
| Solution | Description |
|---|---|
| Reduce the Toxic Effect | Consider using a hemizygous Cre mouse as opposed to using a mouse homozygous for Cre. This will reduce the amount of Cre produced in cells and prevent Cre toxicity while minimizing off-target effects. |
| Use Appropriate Controls | Choice of controls is also crucial. Always use the Cre mouse strain (uncrossed with any floxed mouse line) as a control to determine any unexpected changes in cell number or function. In addition, you could also consider the floxed-only mouse strain as an extra control, to cover all bases. |
| Use Mutant Lox | For experiments involving two or more recombination and/or deletion events, you can use a single Cre to catalyze different reactions. In such cases, make sure to use mutated versions of loxP such as loxN, lox2271 or lox511. In this way, recombination events can occur between the same type of Lox sites, but not between those resulting from different lox sites. |
| Research All Available Resources | When you are all set to acquire your Cre line and Flox line pair, check out commercial vendors that give considerable information about phenotypes and known data on the various strains available. A great resource to check for all mouse knockout studies is the ‘International mouse phenotyping consortium’ (https://www.mousephenotype.org/), which provides detailed phenotypic information as well as vectors, embryonic stem (ES) cells and mouse lines to order for generating your own genetically modified mice. |
What If There Is No Cre or Flox Mouse Available?
- If there is no Cre or Flox mouse line readily available or if you are generating a novel modified mouse line, set aside a considerable amount of time and money and to make your own targeting vectors containing the cre gene under the necessary promoter and the target gene of interest flanked by loxP sites.
- Use these vectors to transfect respective ES cells and select for vector-positive cells by culturing them with antibiotics.
- Test these ES clones for recombination of the vector in the genome by qPCR or southern blot.
- Next, expand these ES clones and microinject into blastocysts from a donor mouse.
- Surgically transfer these blastocysts into a pseudopregnant female mouse and wait for the pups to be born. These will be the chimeric mice that can then be crossed further to obtain pups with the essential transgene in their genome.
- The trick of the trade is to use a different coat colored mouse (agouti, black or white) as the ES cell donor, blastocyst donor, and recipient mother to differentiate between pups that have the genetically modified cells.
Cre-loxP and Beyond
Even with all the limitations and the excessive time and effort that it takes to generate these mouse lines, Cre-loxP systems continue to be one of the most popular genetic tools in multicellular animals and are a mainstay in labs worldwide. Other systems e.g., the Flp-FRT, Dre-rox and ?C31 exist and work similarly to the Cre-lox system. However, the excellent resources available for the cre-lox system make it the most popular technique for genetic modifications in mouse and other lab animals or in vitro cell cultures.
New techniques are emerging and researchers are using older Cre-loxP tools with novel twists to do even more exciting research. With the advent of CRISPR-Cas9 technology, genetic modification using the Cre-loxP system may one day become obsolete. However, until every lab can afford to CRISPR their way through target genes, we have a wonderful spread of Cre-loxP resources at our disposal.
Do not hesitate to give this technology a shot, and if you already use it, share your experience with us!
References
- Jackson laboratories blog post: Cre-Lox myths busted. Sept, 2013.
- Schmidt-Supprian M, Rajewsky K. (2007) Vagaries of conditional gene targeting. Nat Immunol 8(7):665-8.
- Heffner CS, Pratt CH, Babiuk RP, Sharma Y, Rockwood SF, Donahue LR, Eppig JT, Murray SA. (2012) Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource. Nat Commun 3: 1218.
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