The tamoxifen-inducible CreERT2/loxP system is a genetic manipulation tool based on Cre recombinase and a mutant estrogen receptor (ER). This system involves fusing Cre recombinase with a specifically mutated estrogen receptor to form the CreERT2 fusion protein. This mutated estrogen receptor no longer responds to naturally occurring endogenous estrogens but can specifically recognize and bind the exogenous drug tamoxifen.While the standard Cre/loxP system enables gene knockout or expression in specific spatial contexts, the CreERT2/loxP system allows precise temporal control of gene knockout or reporter gene expression upon tamoxifen administration. This technology is crucial for studying the functions of embryonic lethal genes in later developmental stages, conducting cell lineage tracing, and performing mosaic analysis.

(Image: Principle of CreERT System, source: Peking University Laboratory Animal Center)

 

CreERT2 System "Leakage"

 

"Leakage" refers to the phenomenon where the CreERT2 system mediates a certain degree of gene recombination even in the absence of tamoxifen induction. Although the extent of this phenomenon may be low in individual cells, it is sufficient to label a considerable number of cells in various tissues during embryonic development, postnatal development, and adulthood. This non-specific labeling can severely compromise the accuracy of lineage tracing and mosaic analysis experiments, leading to erroneous research conclusions.For example, in the study "Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreERT2 lines," researchers tested three different CreERT2 tool mice (Cdh5-CreERT2, Pdgfb-CreERT2, Prox1-CreERT2) and found that all these strains showed reporter gene activation in control groups without tamoxifen administration. This indicates that the CreERT2 system's ability to completely sequester Cre enzyme in the cytoplasm is not 100% effective, with residual "leakage" allowing random nuclear entry and recombination.

(Image source: Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreERT2 lines)

Additionally, leakage activity may lead to oncogenic transformation of leaky cells, potentially affecting mouse health. Particularly when mice reach older ages, the accumulated number of leaky cells increases, elevating health risks. Therefore, to minimize this risk, it is recommended to conduct experiments when mice are younger. If breeding is necessary, it is best to start mating at 6-8 weeks of age.

 

Differential Sensitivity of Reporter Genes to Basal Activity
 

The sensitivity of reporter genes affects experimental outcomes with the CreERT2 system. Researchers compared the recombination efficiency of four commonly used reporter genes (Ai14, Ai3, mTmG, R26R-EYFP) under the same basal Cre activity. High-sensitivity reporter genes like Ai14 (tdTomato) and Ai3 (EYFP) are easily activated by low levels of basal Cre activity. In contrast, low-sensitivity reporter genes such as mTmG (EGFP) and R26R-EYFP (EYFP) are difficult to activate by basal activity. This difference may be related to the distance between loxP sites—Ai14 and Ai3 have closer loxP sites that facilitate recombination, while mTmG and R26R-EYFP have more distant loxP sites with higher recombination thresholds.

(Image source: Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreERT2 lines)

 

Potential Interference of Basal Activity in Lineage Tracing Experiments
 

Basal activity of the CreERT2 system can also interfere with lineage tracing experiments. For instance, in the brains of mice carrying Cdh5(PAC)-CreERT2 (endothelial cell-specific) and Ai14 reporter genes, in addition to endothelial cells, some labeled microglial cells were observed. Since Cdh5 is not expressed in mature microglial cells, this suggests that these cells may have been labeled by basal Cre activity during early development when their progenitor cells transiently expressed Cdh5. Without strict uninduced control groups, this result could be misinterpreted as endothelial-to-microglial transdifferentiation.

(Image source: Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreERT2 lines)

Experimental Design Recommendations for Cell TracingWhen conducting precise cell tracing experiments, it is essential to recognize the basal activity of the CreERT2 system and the differential sensitivity of reporter genes.
 

Mandatory Control Groups: All experiments using the CreERT2 system must include control groups without tamoxifen administration (genotype: CreERT2/+; Reporter/+) to assess basal activity levels.
 

Careful Selection of Reporter Genes: Choose appropriate reporter genes based on experimental objectives.
 

For lineage tracing, mosaic analysis, and other experiments requiring high precision, prioritize reporter genes with high recombination thresholds, such as mTmG or R26R-EYFP, to minimize background labeling.For experiments requiring bright fluorescent signals for live-cell imaging, Ai14 (tdTomato) remains a good choice due to its brightness and photostability, but its leakage tendency must be acknowledged and results interpreted accordingly.
 

Validation of Experimental Results: In mosaic analysis, reporter gene expression should be closely associated with gene knockout effects. It is best to verify the absence of target proteins through immunohistochemistry or other methods.

 

MingCeler Biotech Animal Center
 

The MingCeler Experimental Animal Research Center operates using a barrier system combined with IVC (Individually Ventilated Cages). The barrier area includes quarantine rooms, clean item receiving rooms, clean item storage rooms, surgical rooms, and three independent housing rooms. Quarantine rooms, surgical rooms, and all three housing rooms are equipped with laminar flow hoods to ensure sterile operating conditions. In terms of biosafety control, personnel access, item access, animal handling procedures, facility equipment management, and early warning mechanisms are governed by relevant operating procedures, with training and strict assessments implemented. Most non-autoclavable items are sent to specialized irradiation companies for sterilization and disinfection. An annual animal quality monitoring plan has been established, clearly defining the conditions for animal access. MingCeler Biotech maintains strict environmental controls and holds comprehensive certifications, including: SPF-grade Experimental Animal Production License, ISO Quality Management System Certification, and full AAALAC accreditation officially approved in October 2025.

 

References:

[1] Álvarez-Aznar, A., Martínez-Corral, I., Daubel, N., Betsholtz, C., Mákinen, T., & Gaengel, K. (2019). Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreERT2 lines. Transgenic Research. https://doi.org/10.1007/s11248-019-00177-8

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