Other useful Cre related resources and databases

Resource Data format Location
Allen Institute for Brain Science Various databases USA
Australian Phenomics Network PhenomBank database Australia
ArrayExpress Database of functional genomics experiments including gene expression where you can query and download data collected to MIAME and MINSEQE standards
Gene Expression Atlas contains a subset of curated and re-annotated Archive data which can be queried for individual gene expression under different biological conditions across experiments
UK
CAGE (Cap-Analysis-Gene-Expression) Library for expression profiling and promoter identification, transcriptional network analysis and transcriptome characterization
Library order: contact@dnaform.jp
Japan
EMAP (Edinburg Mouse Atlas Project) Database of in situ gene expression patterns in the mouse embryo UK
EMMA (The European Mouse Mutant Archive) Cre Recombinase Expressing Strains UK
Eurexpress Transcriptome Atlas Database for Mouse Embryo UK
GENSAT (Gene Expression Nervous System Atlas) List of fully characterized transgenic BAC-Cre recombinase driver lines and database USA
GEO (Gene Expression Omnibus) A public repository that archives and freely distributes microarray, next-generation sequencing, and other forms of high-throughput functional genomic data submitted by the scientific community. In addition to data storage, a collection of web-based interfaces and applications are available to help users query and download the experiments and gene expression patterns stored in GEO USA
IKMC Knockout mouse database Europe
Mouse Resource Browser A resource management project that provides a dynamic and interactive view of 222 world wide available mouse resources, classified in 22 categories Greece
NIH Blueprint Cre driver mice database USA
Pleiades Promoter Project List of completed minipromoters Canada
RIKEN BRC Cre recombinase database Japan
Sanger Mouse Portal A database for mouse research resources at Sanger: BACs, targeting vectors, targeted ES cells, mutant mouse lines, and phenotypic data generated from the Institute's primary screen UK

The Cre/loxP technology allows generation of cell/tissue-selective knock-out models.
This technology is very powerful for studying the function of a gene in a particular biological system and
was successfully used in a very large number of publications. However, scientists that are not familiar with
these recombinase models may misinterpret their results.
These guidelines are written to help the begininners to handle the Cre and CreERT2 mice with care. They are
pointing out the unexpected complexities and technical limitations that can confound the interpretation of
the results.

Before using a Cre line, it is important to check the points below:

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

Circumstances
Cre-driver lines may unexpectedly express the Cre protein either only in male or in female germline or in
both with no particular rule. Penetrance of a generalized Cre mediated deletion in the progeny is much
dependent on the Cre-driver line. It may be a rare or random event that can render this phenomenon difficult
to detect.

Consequences
Experimental group of mutant animals may be heterogenous. Some of the mice will show the expected
recombination pattern whereas total (germline) excision may occur in others. The consequence of this will be
misinterpretation of the data.

Solutions
Always perform genotyping for the deleted allele (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
segregated in the progeny, as there is a risk of maternal contribution, i.e. accumulation of Cre in the
oocyte.

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

Circumstances
Most of the Cre lines are assumed to be undistinguishable from wild-type mice. However it has been shown that
some of them 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; c) toxicity of the Cre recombinase
itself. This Cre toxicity seems to be dependant of the level of expression of the Cre recombinase.
Interestingly, in case of inducible Cre-driver lines (CreERT2), the toxicity appears only after induction
with the Tamoxifen. This strongly suggests that high level of Cre protein causes DNA damage.

Consequences
Misinterpretation of the phenotype observed for the cell/tissue specific knock-out of interest.

Solutions
Preferably phenotype the Cre-driver line before using it to generate conditional knock-outs. In any case 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

Circumstances
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. At first, the expression pattern of a Cre
recombinase driven by a specific promoter may not recapitulate the expression pattern of the corresponding
endogenous gene. Ectopic 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).
Also, it is important to keep in mind that we always observe the end results of a Cre mediated excision. Even
if the Cre-driver line recapitulates the expression pattern of its corresponding endogenous gene, at the time
the mice are analyzed all the daughter cells will show Cre-mediated excision regardless the fact that these
cells continue to express Cre. The end result will be in a much broader and often non expected 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.

Solutions
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.
Beware that in case of inducible Cre-driver line, non-induced deletion (so called ‘leakage’) 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 tamoxifen. 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

Circumstances
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 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 though in the majority of the publications
Tamoxifen has been administrated for very long periods. Infertility, skeletal abnormalities, hepatotoxicity,
behaviour alterations or improved allergic immune response are the principal phenotypes in these studies.
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 advise to use the protocol provided in the annex to minimize Tamoxifen effects. We do not recommend
using Tamoxifen pellets (http://www.innovrsrch.com/) because the duration of delivery of the drug cannot be
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.

  • 1. Non-specificity of Cre-mediated deletion: germline recombination of the
    floxed allele
  • 2. Phenotypes induced by Cre-driver alone: phenotypic characterization of
    Cre-driver line
  • 3. Specificity and efficiency Cre-mediated deletion: Cre-driver and floxed
    target dependency
  • 4. Effects of Tamoxifen treatment: inducible Cre recombinase
  • Summary: recommended experimental procedures when using constitutive or
    inducible Cre drivers:
  • Annex: Protocol for tamoxifen induction of CreER recombination
Choice of Cre reporter line

In all existing Cre reporter lines the expression of a ubiquitous promoter (e.g. ROSA26, CMV, b-actin) drives the production of a reporter protein (e.g. LacZ, GFP etc.) upon the recombination event guided by Cre recombinase. However it should be kept in mind that truly and absolutely ubiquitous promoters (i.e. resulting in expression of a reporter protein at the same level in all the cells of the body) do not exist. E.g. the commonly used Rosa26-LacZ reporter (Soriano, 1999) is known not to be expressed in blood and to be poorly expressed in 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) or green fluorescent protein (GFP) tend to accumulate in mammalian cells. Therefore dynamic view of the recombination/expression events is not possible with these reporters.
 

Promoter Integration site Method of line generation Staining before cre deletion Staining after cre deletion Strain name Direct ordering Original publication Additional data
CMV enhancer/ chicken β-actin ROSA26 HR dimer Tomato (mT) membrane-targeted green fluorescent protein (mG) Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J Jackson Laboratory 17868096 -
CMV enhancer/ chicken β-actin Unknown PNI No staining EGFP B6;D2-Tg(CAG-CAT-EGFP)39Miya  Center for Animal Resources and Development Database 10745079 -
CMV enhancer/ chicken β-actin Unknown PNI No staining β-galactosidase Tg(CAG-cat,-lacZ)34Pva ? 10419695 -
ROSA26 ROSA26 HR No staining EGFP B6;129-Gt(ROSA)26Sortm2Sho/J Jackson Laboratory 11133778 -
CMV enhancer/ chicken β-actin Unknown PNI EGFP Luciferase & β-galactosidase ? ? 20495880 -
ROSA26 ROSA26 HR No staining EYFP B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J Jackson Laboratory 11299042 -
ROSA26 ROSA26 HR No staining ECFP B6.129X1-Gt(ROSA)26Sortm2(ECFP)Cos/Mmnc MMRRC 11299042 -
chicken β-actin Unknown PNI No staining β-galactosidase FVB-Tg(CAG-cat-lacZ)1Brn/StmOrl EMMA 9108159 CreZoo
CMV enhancer/ chicken β-actin ROSA26 HR β-galactosidase EGFP FVB.Cg-Gt(ROSA)26Sortm1(CAG-lacZ,-EGFP)Glh/J Jackson Laboratory 19165827 -
ROSA26 ROSA26 HR No staining tdRFP C57BL/6-Gt(ROSA)26Sortm1Hjf/Ieg EMMA 17171761 -
chicken β-actin Unknown PNI No staining β-galactosidase B6.Cg-Tg(xstpx-lacZ)32And/J Jackson Laboratory 9635195 -
ROSA26 ROSA26 HR No staining β-galactosidase B6.129S4-Gt(ROSA)26Sortm1Sor/J Jackson Laboratory 9916792 CreZoo
ROSA26 ROSA26 HR No staining β-galactosidase B6;129-Gt(ROSA)26Sortm1Sho/J Jackson Laboratory 10220414 -
CMV enhancer/ chicken β-actin Unknown IR β-galactosidase DsRed.T3 Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J Jackson Laboratory 15593332 -
ROSA26 ROSA26 HR No staining Luciferase FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc): A global double-fluorescent Cre reporter mouseKael/J Jackson Laboratory 14717328 -
β-actin Unknown PNI GFP Luciferase Tg(Actb-GFP,-luc)#Nki ? 14612492 -
CMV enhancer/ chicken β-actin Unknown PNI DsRed-Express EGFP B6.Cg-Tg(CAG-DsRed,-EGFP)5Gae/J Jackson Laboratory 18543298 -
CMV enhancer/ chicken β-actin Unknown IR β-galactosidase Alkaline phosphatase Tg(CAG-Bgeo/ALPP)1Lbe/J Jackson Laboratory 10191045 CreZoo
CMV enhancer/ chicken β-actin Unknown IR β-galactosidase EGFP B6.129(Cg)-Tg(CAG-Bgeo/GFP)21Lbe/J Jackson Laboratory 11105057 -
CMV enhancer/ chicken β-actin ROSA26 HR No staining β-galactosidase FVB.Cg-Gt(ROSA)26Sortm1(CAG-lacZ,-EGFP)Glh/J Jackson Laboratory 19165827 -
CMV enhancer/ chicken β-actin ROSA26 HR No staining EGFP FVB.Cg-Gt(ROSA)26Sortm1(CAG-lacZ,-EGFP)Glh/J Jackson Laboratory 19165827 -
CMV enhancer/ chicken β-actin ROSA26 HR No staining EYFP B6.Cg-Gt(ROSA)26Sortm3(CAG-EYFP)Hze/J Jackson Laboratory 20023653 -
CMV enhancer/ chicken β-actin ROSA26 HR No staining tdTomato B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J Jackson Laboratory 20023653 -
CMV enhancer/ chicken β-actin ROSA26 HR No staining ZsGreen1 B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J Jackson Laboratory 20023653 -
Promoter Integration site Method of line generation Staining before cre deletion Staining after cre deletion Strain name Direct ordering Original publication Additional data
CMV enhancer/ chicken β-actin ROSA26 HR β-galactosidase EGFP FVB.Cg-Gt(ROSA)26Sortm1(CAG-lacZ,-EGFP)Glh/J Jackson Laboratory 19165827 -
ROSA26 ROSA26 HR No staining alkaline phosphatase B6;129S4-Gt(ROSA)26Sortm2Dym/J Jackson Laboratory 11687793 -
Promoter Integration site Method of line generation Staining before cre deletion Staining after cre deletion Strain name Direct ordering Original publication Additional data
ROSA26 ROSA26 HR No staining β-galactosidase ? ? 19692579 -
Methods of line generation
HR: the genetically modified mouse line was generated by
gene targeting (homologous recombination in ES cell followed by injection of these modified ES cells in
blastocysts to derive a mouse line)
IR: the genetically modified mouse line was generated by
random integration of a DNA construction in ES cell followed by injection of these modified ES cells in
blastocysts to derive a mouse line
PNI: transgenic mouse was generated by micro-injection of
a DNA construction in the pronucleus (PNI transgenesis)