1. Non-specificity of Cre-mediated deletion: germline recombination of the floxed allele

Concern

Cre-driver lines may unexpectedly express the Cre protein in the germlines of male, female, or both. Penetrance of a generalized Cre-mediated deletion in the progeny is dependent on the Cre-driver line. Thus, it may be a rare event making this phenomenon difficult to detect.

Consequences

Experimental groups of mutant animals may be genetically heterogeneous. Some of the mice will show the expected recombination pattern while complete (germline) excision would occur in others. The consequence of this will be misinterpretation of the data.

Solution

Always perform genotyping for the deleted allele on an untargeted organ (e.g. PCR for the knock-out allele on the tail lysates). All the pups generated from a breeding with a Cre-driver line should be tested, even if the Cre transgene is not present in the progeny. This will demonstrate the frequency of germline Cre expression and resolve the potential problem of maternal contribution, i.e. Cre from an oocyte persisting in the early embryo.

2. Phenotypes induced by Cre-driver alone: phenotypic characterization of Cre-driver line

Concern

Most of the Cre lines are assumed to be undistinguishable from wild-type mice. However it has been shown that some can generate a phenotype on their own. This may be caused by: 1) method of generation of the line (e.g. more frequently observed with transgenics generated by random insertion after pronuclear injection); 2) presence of passenger genes carried by modified BACs; or 3) toxicity of the Cre recombinase itself. This Cre toxicity seems to be dependant of the level of expression of the Cre recombinase. In case of inducible Cre-driver lines (e.g. CreERT2 tamoxifen inducible Cre), the toxicity appears only after induction.

Consequences

Misinterpretation of the phenotype observed for the cell/tissue specific knock-out of interest. Since high levels of Cre protein cause DNA damage, it will commonly appear as a DNA-damage phenotype.

Solution

The preferable course is to phenotype the Cre-driver line before using it to generate any conditional knock-outs. If this is not possible, you must use the Cre-driver as a control group instead of (or along with) wild type mice in your experiments. Make sure this line is on the same genetic background as your conditional knock-out mice to avoid any strain-specific phenotype.

3. Specificity and efficiency Cre-mediated deletion: Cre-driver and floxed target dependency

Concern

Ideally, the expression pattern of a Cre-driver line should be restricted to a specific cell type or tissue. In some cases, the observed expression pattern is more complex. Firstly, the expression pattern of a Cre recombinase driven by a specific promoter may not recapitulate the expression pattern of the corresponding endogenous gene. “Leaky” expression might be observed, especially if the line was generated by pronuclear injection (due to insertion of the transgene at the proximity of enhancers, suppressors and other regulatory elements). Secondly, it is also important to closely examine the end results of a Cre mediated excision. Each floxed locus will have its own innate susceptibility to Cre recombination, based on nuclear architecture, transcriptional activity, etc. Even if the Cre-driver line recapitulates the expression pattern of its corresponding endogenous gene, you must check the resulting effect on your floxed locus. Finally, Cre protein expression in development may not reflect the adult expression. If an earlier cell progenitor expresses Cre, it will expand the number and of cells affected. Do not assume that Cre expression in the adult is the tissue-specificity throughout development. This phenomenon can result in a much broader and often unexpected deletion pattern. It should be also kept in mind that the efficiency of recombination induced by a specific Cre-driver is highly dependent of the floxed allele that is used.

Consequences

Unexpected (non-specific, broader or more restricted) pattern of recombination that leads to misinterpretation of the results.

Solution

The pattern and efficiency of Cre-mediated excision must be evaluated for each floxed allele even if the Cre-driver is well characterized. Analysis can be performed by qPCR or Southern Blot. Ideally, where possible, in situ analysis of tissue for target protein or RNA should be performed. Beware that in case of inducible Cre-driver line, non-induced deletion may occur. Therefore each inducible Cre-driver line should be assessed for leakage, i.e. for the ability to cause Cre mediated excision in the absence of induction. Pattern and efficiency of Cre-mediated excision must be evaluated for each animal. If leakage is observed, the non-induced cohort cannot be considered as a control group.

4. Effects of Tamoxifen treatment: inducible Cre recombinase

Concern

One of the most widely used strategies for inducing temporally specific Cre activity involves fusing the Cre recombinase with the mutated ligand-binding domain (ER or ERT2) of the human estrogen receptor therefore producing chimeric recombinases, e.g. CreER or CreERT2. ER and ERT2 domains have low affinity to endogenous estrogens allowing the chimeric Cre recombinases to remain cytoplasmic in untreated animals. The ER or ERT2 domains are activated by synthetic estrogen receptor antagonist drugs Tamoxifen (or 4-OH tamoxifen). Upon activation CreER or CreERT2 is able to translocate from the cytoplasm to the nucleus and induce the recombination of floxed alleles. The effects of Tamoxifen in the mouse are quite well documented in the literature. Tamoxifen has been administrated for very long periods. Infertility, skeletal abnormalities, hepatotoxicity. behaviour alterations or improved allergic immune response are the principal effects seen. Severe gonadic phenotypes are also observed when Tamoxifen was administrated neonatally or prenatally.

Consequences

Complication of observed conditional knock-out phenotypes by phenotypes induced by Tamoxifen administration. Studies lacking evaluation of Tamoxifen induced phenotypes may be questioned.

Solution

Always include a group of Tamoxifen treated wild type mice in all the experiments involving inducible Cre drivers. Ideally these animals should be on the same genetic background as the cell/tissues specific gene ablated mice to avoid any strain specific phenotype. We also advise use of the protocol provided in the annex to minimize Tamoxifen effects by limiting duration of exposure. We do not recommend using Tamoxifen pellets (www.innovrsrch.com) because the duration of delivery of the drug cannot be as easily controlled. Administration of Tamoxifen by intraperitoneal injection or oral gavage for 5 days followed by a latency period of 4 weeks before starting experiments is an efficient procedure to reduce Tamoxifen induced phenotypes.

Summary: recommended experimental procedures when using constitutive or inducible Cre drivers

1. Systematic genotyping for presence of the deleted allele during breeding steps
2. If you are using a constitutive Cre: use Cre animals as controls
3. If you are using a inducible Cre: use CreERT2 animals induced with Tamoxifen and wild type animals induced with Tamoxifen as controls
4. Efficiency of Cre-mediated excision should to be checked for each floxed allele and in all the animals of the experimental groups.

Choice of Cre reporter line

Cre-reporter lines are designed to have Cre dependent expression of a transgenic marker gene, usually LacZ or GFP, to visualize the range of expression of a Cre recombinase in a particular Cre line. All existing Cre reporter lines use a ubiquitous promoter, e.g. ROSA26, CMV, b-actin, to drive production of the marker by Cre recombinase. It should be kept in mind that true ubiquitous promoters (i.e. resulting in equal levels of expression all the cells of the body) do not exist. The commonly used Rosa26-LacZ reporter (Soriano, 1999), for example, is known to not to express in blood and to express only poorly in the brain. Thus, scientists are advised to verify if the chosen reporter line is indeed expressing the reporter protein at a sufficient level in each cell type of interest in the time course of their study (e.g. by crossing the line to a known strong ubiquitous Cre driver line).

Unspecific reporter signal

In the mouse, endogenous β-galactosidase activity can often lead in false-positive results. Endogenous autofluorescence is also common issue when using fluorescent protein reporters (GFP, YFP etc.). Wild type controls are indispensable for detection of these false positives.

Accumulation of the reporter protein

β-galactosidase (LacZ) and green fluorescent protein (GFP) tend to accumulate in mammalian cells. Therefore dynamic view of the recombination/expression events through development is often not possible with these reporters.