Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice

Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreERTm and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1PB-CreERTm;R26RlacZ mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs

Tamoxifen-induced Cre subcellular localization is time-dependent.

<p>Representative islets from adult <i>Pdx1^PB</i>-<i>CreER^Tm</i>;<i>Vegfa^loxP</i> mice given 8 mg tamoxifen (Tm, <b>D–L</b>) or corn oil vehicle (<b>A–C</b>) in three subcutaneous injections. Pancreata were harvested 1 week (<b>D–F</b>), 1 month (<b>G–I</b>), or 3 months (<b>J–L</b>) following the last injection and labeled with antibodies against insulin (green; <b>A</b>, <b>D</b>, <b>G</b>, <b>J</b>) and Cre recombinase (red; <b>B</b>, <b>E</b>, <b>H</b>, <b>K</b>). Merged images are shown in <b>C</b>, <b>F</b>, <b>I</b>, <b>L</b>. Scale bar in <b>A</b> is 50 µm, and applies to panels <b>B–L</b>.</p

Duration of tamoxifen-induced gene recombination is dose-dependent.

<p>The percentage of insulin+ β-cells expressing β-gal in islets from pancreas sections (<b>A</b>) and from transplanted islet grafts (<b>B</b>) is shown. Each data point represents the percentage of double-positive cells to all insulin+ β-cells counted in a single mouse sample. Cre−, <i>R26R^lacZ</i> control islets; Cre+, <i>Pdx1^PB</i>-<i>CreER^Tm</i>;<i>R26R^lacZ</i> islets; Tm, tamoxifen. <b>C</b>. Amount of recombination observed in islet grafts from mice given either 3×1 mg or 3×8 mg tamoxifen at the indicated time points following the last tamoxifen injection. Data in <b>C</b> is expressed as means of data in <b>B</b>.</p

Higher dose tamoxifen induces recombination weeks following administration.

<p><b>A.</b> Islets from untreated <i>Pdx1^PB</i>-<i>CreER^Tm</i>;<i>R26R^lacZ</i> mice or <i>R26R^lacZ</i> controls were transplanted into mice given three subcutaneous injections of 8 mg tamoxifen (Tm) or corn oil vehicle at the indicated times following the last injection. Pancreata and islet grafts were harvested 2 weeks after the final injection. <b>B–C.</b> Representative pancreatic islets from <i>R26R^lacZ</i> (Cre−) mice (<b>B</b>) or <i>Pdx1^PB</i>-<i>CreER^Tm</i>;<i>R26R^lacZ</i> (Cre+) mice (<b>C</b>) treated with 3×8 mg Tm. Cryosections were labeled with antibodies to insulin (green; <b>B</b>, <b>C</b>) and β-galactosidase (β-gal, red; <b>B</b>, <b>B′</b>, <b>C</b>, <b>C′</b>). Scale bar in <b>B</b> is 50 µm, and applies to panels <b>B′</b>, <b>C</b>, <b>and C′</b>. <b>D–I</b>. Islet graft cryosections were labeled with antibodies to insulin (green; <b>D–I</b>) and β-gal (red; <b>D–I</b>, <b>D′–I′</b>). Scale bar in <b>D</b> is 50 µm, and applies to panels <b>E–I</b>, <b>D′–I′</b>. <b>J–O</b>. β-gal activity was tested in islet grafts using X-gal. Scale bar in <b>J</b> is 100 µm, and applies to panels <b>K–O</b>. Images of the full graft cross-sections (before cropping and rotating for visual clarity) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033529#pone.0033529.s004" target="_blank">Figures S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033529#pone.0033529.s005" target="_blank">S5</a>.</p