Tamoxifen-independent recombination in the RIP-CreER mouse

Abstract

Background: The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas.

Principal findings: Here, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains.

Significance: Evidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic β-cells.

Figure 1. Tamoxifen-independent expression of HA3-ICA512-CCF in β-cells of RIP-CreER, R26-Stop-HA3-ICA512-CCF mice.

A. Strategy for the generation of the inducible R26-Stop-HA3-ICA512-CCF knock-in mouse line. B. Western blotting of pancreatic islet extracts from 3-month-old tamoxifen-treated or untreated RIP-CreER, R26-Stop-HA3-ICA512-CCF mice or tamoxifen-treated R26-Stop-HA3-ICA512-CCF mice with anti-HA and anti-γ-tubulin antibodies (similar results were obtained from 3 independent experiments). C-N. Confocal microscopy images of pancreatic cryosections from 4-month-old tamoxifen-treated RIP-CreER, R26-Stop-HA3-ICA512-CCF (C-F) and R26-Stop-HA3-ICA512-CCF (G-J) and untreated RIP-CreER, R26-Stop-HA3-ICA512-CCF (K-N) mice stained with DAPI (C, G and K), as well as with anti-insulin (D, H and L) and anti-HA antibodies (E, I and M). Triple stainings (merge) are shown in F, J and N. Scale bars in F, J and N equal 10 µm. O. Quantification and statistical analysis of HA3-ICA512-CCF-positive cells relative to total insulin-positive cells in tamoxifen-treated or untreated RIP-CreER, R26-Stop-HA3-ICA512-CCF mice, and tamoxifen-treated R26-Stop-HA3-ICA512-CCF mice. Dots represent individual islets from 3 mice in each genotype group. The p value of a t-test between groups is shown.

Figure 2. β-galactosidase gene (lacZ) expression in RIP-CreER, Rosa26-lacZ mice.

A-C. LacZ staining of the pancreatic tissue from 10-week-old tamoxifen-treated RIP-CreER, Rosa26-lacZ mice (A), and Rosa26-lacZ (B) and untreated RIP-CreER, Rosa26-lacZ (C) mice. D. Quantification and statistical analysis of the lacZ-positive area relative to the total islet area in tamoxifen-treated or untreated RIP-CreER, Rosa26-lacZ mice, and tamoxifen-treated Rosa26-lacZ mice. Dots represent individual islets from 3 mice in each genotype group. The p value of a t-test between groups is shown.

Figure 3. Tamoxifen-independent knockout of PTBP1 in adult β-cells of RIP-CreER, PTBP1^loxP/loxP mice.

A-L. Confocal microscopy images of pancreatic cryosections from 8-week-old tamoxifen-treated RIP-CreER, PTBP1^loxP/loxP (A-D) and PTBP1^loxP/loxP (E-H) and untreated RIP-CreER, PTBP1^loxP/loxP (I-L) mice stained with DAPI (A, E and I), as well as with anti-PTBP1 (B, F and J) and anti-insulin antibodies (C, G and K). Triple stainings (merge) are shown in D, H and L. Scale bars in D, H and L equal 20 µm. M. Quantification and statistical analysis of PTBP1-positive cells relative to total insulin-positive cells in tamoxifen-treated and untreated RIP-CreER, PTBP1^loxP/loxP mice, and tamoxifen-treated PTBP1^loxP/loxP mice. Dots represent individual islets from 4 mice in each genotype group. The p value of a t-test between groups is shown.

Figure 4. β-galactosidase gene (lacZ) expression in Pdx1-CreER, Rosa26-lacZ mice.

A-C. LacZ staining of the pancreatic tissue from 4-month-old tamoxifen-treated Pdx1-CreER, Rosa26-lacZ mice (A), and Rosa26-lacZ (B) and untreated Pdx1-CreER, Rosa26-lacZ (C) mice. D. Quantification and statistical analysis of the lacZ-positive area relative to the total islet area in tamoxifen-treated or untreated Pdx1-CreER, Rosa26-lacZ mice, and tamoxifen-treated Rosa26-lacZ mice. Dots represent individual islets from 3 mice in each genotype group. The p value of a t-test between groups is shown.