The Cre-lox system is one of the most revolutionary tools in the geneticist's toolbox. You don't have to be a user or developer of cre or floxed mice to appreciate the impact that cre-lox technology has had on biomedical research and genetic engineering. Since its discovery in the early 1980's and its subsequent use in the mouse in the early 1990's, it has become known as the most sophisticated method of manipulating the mouse genome. Despite its widespread use, Cre-lox strains may not behave exactly as some researchers expect, and they can be used in some very creative ways.
1. It can matter if Cre is transmitted from the female or the male parent
Cre activity can vary depending if transgene is inherited from the male or female parent. For example, EIIa-Cre, B6.FVB-Tg(EIIa-cre)C5379Lmgd/J (003724), converts floxed alleles to complete knockout alleles because Cre is expressed throughout the mouse, but it is significantly more efficient when transmitted maternally. The Camk2a-Cre, B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (005359), recombine sequences in the hippocampus, but there is expression in male germ cells as well. The parental inheritance pattern may not matter for all Cre strains, but it is a good idea to compare results obtained from both maternal and paternal transmission whenever possible.
2. Cre alone can produce a phenotype
Cre is often expressed from a randomly integrated transgene, but very few insertion sites are known. Random transgene integration can disrupt an endogenous mouse gene and cause unanticipated side effects. Since hemizygous mice have a wildtype chromosome present, using hemizygous instead of homozygous Cre genotypes might minimize unintended consequences of random transgene insertion. Cre toxicity can also occur when expression is high enough to affect cell physiology, or when Cre recognizes genomic sequences that resemble loxP sites. Cre can damage DNA in fibroblasts, gastric cells, and sperm in the absence of any floxed sequence. For these reasons, it is wise to include the Cre mouse itself (without any floxed sequences) as a control group in your experiments.
3. “Ubiquitous” Cre strains are not always as ubiquitous as you might think
The tamoxifen-inducible Cre strain, B6.Cg-Tg(CAG-cre/Esr1)5Amc/J (004682), is used to conditionally delete floxed sequences throughout the entire mouse because its promoter (beta actin) should have ubiquitous expression. Data collected by the JAX Cre Resource show that tamoxifen induction is more robust in certain tissues (pancreas and smooth muscle, for example) than others (spleen and ovary). Differences in recombination efficiency among tissue types were also noted in a similar strain, B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J (008085).
4. There is a way to convert a floxed allele to a knockout, even if the mouse doesn’t carry a Cre transgene
Oocytes in Vasa-Cre strains, FVB-Tg(Ddx4-cre)1Dcas/J (006954) and B6.FVB-Tg(Ddx4-cre)1Dcas/KnwJ (018980), express Cre mRNA/protein even when the oocyte itself does not carry the transgene. Strains with maternal Cre expression in the oocyte can save time when converting floxed alleles to complete knockouts; no further breeding is needed to remove the Cre transgene after confirming that recombination took place. Other strains with similar characteristics include the Sox2-cre strains: 008454, 014094, and 004783.
5. There are tools to understand the level of Cre activity
There is a Cre reporter, B6.129P2-Gt(ROSA)26Sortm3Nik/J (013587), that up-regulates both a red and a green fluorescent protein in cells with Cre activity. When Cre activity is especially high, however, the green fluorescence is silenced. This tool can be used to understand the relative intensity of Cre activity in different cells or tissues.
6. Cre can be linked to the Tet system, and vice versa
The Tet system provides control over the timing and location of gene expression using two transgenes: a tetracycline-dependent transcription factor (tTA or rtTA) that determines tissue specificity, and a responsive promoter (tetO) that expresses the transgene of interest. But what if you don’t have a tTA or rtTA transgenic mouse that targets your cell type, but you do have a Cre mouse with the right specificity? Mating Cre to B6.129P2(Cg)-Gt(ROSA)26Sortm1(tTA)Roos/J (011008) or B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J (005670) will give you tTA or rtTA expression, respectively, in the right tissues. Similarly, there is a tetO-Cre transgene available on C57BL/6 and BALB/c genetic backgrounds that expresses Cre if you already have a tTA or rtTA strain for your cell type.
7. Cre might not be active in every cell you expect
“Cre mosaicism” is documented in some strains, resulting in incomplete recombination in all cells. Data from the JAX Cre Resource have documented this in several examples.
8. You should not need a homozygous Cre transgene for efficient recombination
Expression levels from a hemizygous transgene should be enough to see recombination. Randomly integrated transgenes can lead to surprising phenotypes when they are bred to homozygosity.
9. Cre-lox can be used to create new tissue- or cell type- specific fluorescent strains
Do you need to fluorescently label a cell type, but can’t find the right transgenic mouse? If you have a Cre mouse that targets the cells you’re interested in, you can mate it with a Cre reporter to get red, green, yellow, or even rainbow-colored fluorescence in those cells. If you prefer histology, other Cre reporters can give you beta-galactosidase (LacZ), alkaline phosphatase, or luciferase expression.
10. Some floxed alleles are more easily recombined than others
If the Cre-lox system is incompletely knocking out a particular gene, remember that some genes are more easily recombined than others, possibly because the chromatin state is preventing Cre from accessing the loxP sites.
11. Inducible Cre can be leaky
Cre is sequestered in the cytoplasm when it is fused to the ligand binding domain of the estrogen receptor, and enters the nucleus when exposed to tamoxifen. It is generally assumed that Cre is excluded from the nucleus (and thereby unable to recombine loxP sites) in the absence of tamoxifen, but there are examples of a ubiquitous Cre, a pancreas-specific Cre and a melanocyte-specifc Cre recombining sequences without induction.
12. The Jackson Laboratory has many resources to help you with Cre-Lox
The JAX Cre Resource is a great source of information about many strains available from The Jackson Laboratory. The Cre Portal can help you find Cre mice for your research. Not sure if your Cre is active when and where you expect it? A Cre reporter can help. Still have questions? Technical Information Services can help you find strains or answer your lingering questions about Cre-Lox.