H11-CAG-MerCreMer Mice

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Catalog Number: C001356

Strain Name: C57BL/6JCya-Igs2em1(CAG-MerCreMer)/Cya

Genetic Background: C57BL/6JCya

Reproduction: Carriers x Carriers


Strain Description

The CAG promoter is an artificially constructed combination promoter consisting of the cytomegalovirus (CMV) early enhancer element and the chicken beta-actin promoter, commonly used to drive high-level expression of genes in expression vectors in mammals.

The H11-CAG-MerCreMer mouse is a tamoxifen-inducible tool mouse that ubiquitously expresses the MerCreMer recombinase under the control of the CAG promoter. Compared to Cre recombinase fused with a single estrogen receptor (CreMer or CreERT), the double ER-fusion Cre recombinase (MerCreMer) exhibits higher Cre recombinase activity and less expression leakage. In the absence of tamoxifen, MerCreMer recombinase remains in the cytoplasm. Upon tamoxifen treatment, the recombinase translocates into the nucleus to exert its recombination function. When crossed with mice harboring loxP sites, the offspring will undergo Cre-mediated recombination between loxP sites in a wide range of cells and tissues after tamoxifen induction. This strain is homozygous viable.

Strain Strategy

The CAG-MerCreMer gene expression cassette was integrated into the H11 safe harbor locus of the mouse genome through gene-editing techniques.


Applications

This tamoxifen-inducible mouse can serve as a Cre recombinase tool for systemic loxP recombination. For example, crossing this mouse with conditional knockout mice allows for systemic deletion of sequences between two loxP sites.

Induction Protocol Exploration

  • (1) Detection methods


    Experimental group (TAM+)

    Cohorts of 8–14-week-old CAG-MerCreMer[KI/+];Rosa26-LSL-tdTomato[CKI/+] mice were subjected to tamoxifen induction via intraperitoneal injection or oral gavage. Tamoxifen was administered for a total of five doses, with intervals of 24 or 48 hours, at varying concentrations: 1 mg/mouse, 2 mg/mouse, 0.25 mg/mouse, 0.5 mg/mouse, 0.75 mg/mouse, or 12.5 mg/kg, 25 mg/kg, 50 mg/kg, and 100 mg/kg.

  • Control group (TAM-)

    Age-matched CAG-MerCreMer[KI/+];Rosa26-LSL-tdTomato[CKI/+] mice were treated with an equivalent volume of corn oil via intraperitoneal injection for five doses, with intervals of either 24 or 48 hours.

  • Negative Control Group (TAM+ Blank)

    Cohorts of Rosa26-LSL-tdTomato[CKI/CKI] mice were treated with tamoxifen at doses of 1 mg/mouse or 0.75 mg/mouse for five consecutive administrations, either by intraperitoneal injection or oral gavage, with 24- or 48-hour intervals.

  • Outcome: Survival curves were generated starting from the time of the first injection to monitor the mortality rates across experimental groups.

    • (2) Results


    • Figure 1. Survival curves of CAG-MerCreMer[KI/+];Rosa26-LSL-tdTomato[CKI/+] mice following induction with varying doses of tamoxifen.

    • a. In the experimental group (TAM+), CAG-MerCreMer[KI/+];Rosa26-LSL-tdTomato[CKI/+] mice received intraperitoneal injections (i.p.) of tamoxifen at 1 mg/mouse every 24 hours, leading to 100% mortality by day 6 post-induction. Increasing the tamoxifen dose to 2 mg/mouse accelerated mortality, with 100% of mice succumbing by day 4. In contrast, mice in the negative control group (TAM+ Blank), which received 1 mg/mouse tamoxifen via i.p. injection, showed no mortality, and similarly, no mortality was observed in the control group (TAM-) treated with equivalent volumes of corn oil. These data suggest that tamoxifen doses of 1 mg/mouse and 2 mg/mouse induce high mortality, rendering these doses unsuitable for subsequent induction studies.

      b. In the experimental group (TAM+), administering tamoxifen at 0.25 mg/mouse and 0.5 mg/mouse via i.p. injection at 24-hour intervals led to mortality by day 7 post-induction, while a dose of 0.75 mg/mouse resulted in mortality by day 8. When 0.75 mg/mouse tamoxifen was administered via intragastric (i.g.) route, mortality was delayed until day 10 post-induction. These findings indicate that mortality rates are relatively consistent within the 0.25–0.75 mg/mouse dose range, with small variations in lethality. Nevertheless, all doses tested—0.25, 0.5, and 0.75 mg/mouse—resulted in eventual mortality, further emphasizing the unsuitability of these doses for induction experiments.

      c. In the experimental group (TAM+), administering 100 mg/kg tamoxifen i.p. at 24-hour intervals led to 100% mortality by day 3, while extending the interval to 48 hours delayed 100% mortality until day 6. At a dose of 50 mg/kg, 100% mortality occurred by day 7 with 24-hour intervals, and by day 7 with 66.6% mortality when dosed at 48-hour intervals. For 25 mg/kg tamoxifen, mortality was observed by day 14 with 24-hour intervals, but no mortality was observed at 48-hour intervals. Importantly, no mortality was noted in mice receiving 12.5 mg/kg tamoxifen, regardless of the interval (24-hour or 48-hour). These results demonstrate a clear dose-dependent relationship between tamoxifen administration and mortality rates in CAG-MerCreMer[KI/+]; Rosa26-LSL-tdTomato[CKI/+] mice, with increasing tamoxifen concentrations correlating with higher mortality. Extending the interval between doses mitigates mortality, suggesting that both dose and interval must be carefully optimized to balance induction efficacy with animal survival.

    • Recommended Induction Protocol and Sampling Time

    • For optimal results, we recommend induction with 12.5 mg/kg tamoxifen administered at 24-hour intervals for a total of 5 doses. Tissue collection should be performed 7 days post-induction to achieve maximal efficiency.

    • Validation Data

    • (1) Methodology

    • H11-CAG-MerCreMer mice were crossed with Rosa26-LSL-tdTomato mice to generate heterozygous offspring expressing both MerCreMer recombinase and the LSL-tdTomato fluorescent reporter. Following tamoxifen induction, Cre recombinase efficiently excises the stop element (LSL) upstream of the tdTomato sequence, resulting in the expression of the tdTomato fluorescent protein in Cre-positive cells. Peripheral blood was collected 7 days post-induction for flow cytometry (FACS) analysis to assess tdTomato expression. Additionally, tissues from the lung, liver, brain, spleen, kidney, stomach, heart, and rectum were harvested for fluorescence microscopy, enabling the evaluation of Cre recombinase activity across various tissues.

    • (2) Experimental groups

    • Cre+Tam+: CAG-MerCreMer[KI/+]; Rosa26-LSL-tdTomato[CKI/+] treated with tamoxifen.

      Cre+Tam-: CAG-MerCreMer[KI/+]; Rosa26-LSL-tdTomato[CKI/+] treated with corn oil.

      Cre-Tam+: Rosa26-LSL-tdTomato[CKI/CKI] treated with tamoxifen.

    • (3) Results

    • a. Cre recombinase expression in peripheral blood immune cells


    • Figure 2. FACS analysis of Cre recombinase-mediated recombination in peripheral blood immune cells.

    • ①: Robust tdTomato expression was detected in peripheral blood CD45+CD11b+ cells, with a recombination efficiency of 99.68%. In CD45+CD3+ T cells, the recombination efficiency was 85.54%; in CD45+CD19+ B cells, it was 75.13%; and in CD3-CD335+ NK cells, the efficiency reached approximately 96.16%.

      ②: Recombination efficiency in CD45+CD11b+ cells was 98.95%, while in T cells it was 63.38%, in B cells 66.76%, and in NK cells approximately 91.05%.

      ③: CD45+CD11b+ cells showed a recombination efficiency of 99.6%. In T cells, the efficiency was 76.61%, in B cells 89.89%, and in NK cells around 89.52%.

      ④: Recombination efficiency in CD45+CD11b+ cells was 98.79%, while in T cells it was 64.05%, in B cells 90.22%, and in NK cells approximately 86.27%.

      ⑤~⑥: In the corn oil-treated control group (Cre+Tam-), minimal tdTomato expression leakage was observed in peripheral blood immune cells, whereas in the Cre-Tam+ group, leakage was nearly undetectable.

    • b. Cre recombinase expression in the lung and liver


    • Figure 3. Autofluorescence of tdTomato protein expression in the lungs and liver mediated by Cre recombinase.       In Cre+Tam+ mice, strong orange autofluorescence was observed in the alveoli, bronchial epithelial cells, smooth muscle fibers, and liver tissues, indicating effective Cre-mediated recombination in these cell types. In the control group (Cre+Tam-), minimal expression leakage was detected in the lung, with some leakage observed in the liver.

    • c. Cre recombinase expression in the brain and spleen


    • Figure 4. Autofluorescence of tdTomato protein expression in the brain and spleen mediated by Cre recombinase.       In Cre+Tam+ mice, partial orange autofluorescence was detected in the third ventricle, fourth ventricle, olfactory bulb, and cerebellum of the brain. In the spleen, robust orange autofluorescence was seen in the white pulp, red pulp, and trabeculae, reflecting Cre recombination in these tissues.

    • d. Cre recombinase expression in the kidneys and stomach


    • Figure 5. Autofluorescence of tdTomato protein expression in the kidneys and stomach mediated by Cre recombinase.       In Cre+Tam+ mice, significant orange autofluorescence was observed in the tubular epithelial cells and glomeruli of the kidneys, as well as in the mucosal and muscular layers of the stomach, indicating successful recombination in these tissues. Minimal expression leakage was noted in the stomach tissues of Cre+Tam- control mice.

    • e. Cre recombinase expression in the heart and rectum


    • Figure 6. Autofluorescence of tdTomato protein expression in the heart and rectum mediated by Cre recombinase.       In Cre+Tam+ mice, significant orange autofluorescence was detected in the heart tissues, with autofluorescence signals also evident in the rectum, indicating Cre recombination in these regions.

    • (4) Conclusion

    • In H11-CAG-MerCreMer mice treated with tamoxifen, substantial Cre recombinase activity was observed in peripheral blood CD45+CD11b+, CD45+CD3+, CD45+CD19+, and CD45+CD335+ cells. Strong fluorescent signals indicating successful recombination were also detected in the lungs, liver, spleen, kidneys, stomach, and heart, with partial expression seen in the brain and rectum. Based on these findings, we recommend a tamoxifen induction protocol of 12.5 mg/kg at 24-hour intervals for 5 doses, followed by tissue collection 7 days post-induction to achieve optimal results.

    • This model may have an elevated sensitivity to tamoxifen induction. We recommend customizing the induction protocol and conducting pilot studies to minimize risks. If the target gene impacts survival or phenotype, consult relevant literature and tailor induction protocols accordingly. Validation results are for reference and may vary across studies.

    • Announcements

    • The MerCreMer recombinase gene expression cassette is integrated into chromosome 11 in this strain. It is recommended that breeding with Flox mice targeted on the same chromosome is avoided.