Cre-loxP Recombination Essentials Part 2

The Cre-loxP recombination system is routinely used for the generation of mouse knockouts. In part 1 of this mini-series, I introduced the concept and applications of Cre-loxP. 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 take you through some of these pitfalls along with potential solutions to solve your Cre-loxP challenges.1

Common Pitfalls With the Cre-loxP System:

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

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’ (, 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?

  1. 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.
  2. Use these vectors to transfect respective ES cells and select for vector-positive cells by culturing them with antibiotics.
  3. Test these ES clones for recombination of the vector in the genome by qPCR or southern blot.
  4. Next, expand these ES clones and microinject into blastocysts from a donor mouse.
  5. 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.
  6. 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 φC31 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!


  1. Jackson laboratories blog post: Cre-Lox myths busted. Sept, 2013.
  2. Schmidt-Supprian M, Rajewsky K. (2007) Vagaries of conditional gene targeting. Nat Immunol 8(7):665-8.
  3. 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 resourceNat Commun 3: 1218.
Image credit: Jake Rustenhoven

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