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. 2022 Feb 3;106(6):1083–1097. doi: 10.1093/biolre/ioac029

Figure 1.

Figure 1

(A) A schematic describing the Cre-Lox system utilized in our model. In the CAG-Cre-Esr1 mice, the Cre recombinase is fused with a mutated LBD of the ER, which has very high affinity to the synthetic ER ligand tamoxifen while lacking sensitivity to endogenous estrogen. Upon tamoxifen administration, Cre recombinase translocates to the nucleus where it excises exon 2 of the CXCR4 gene flanked by the LoxP sites. (B) A schematic of the experimental model. WT C57BL/6 J recipient female mice received nongonadotoxic submyeloablative BMT regimen using 5-FU injections on day −6 and day −1 before BMT and SCF injections at −21 and −9 h prior to and +3 h after the second 5FU dose. Cre+CXCR4flox/flox/GFP+ (CXCR4KO) or Cre−CXCR4flox/flox/GFP+ (WT) mice served as BM donors. BM engraftment was assayed by flow cytometry of peripheral blood on day 21 post-BMT. Subsequently, engrafted female mice received intraperitoneal injection of Tamoxifen at 75 mg/kg body weight for five consecutive days. BMT recipients were mated with fertile males on day 14 post-tamoxifen injections and timed pregnancies were established. Pregnant mice were sacrificed on E9.5 or as nonpregnant (controls). Blood, BM, spleen, and uterus were collected from pregnant and nonpregnant controls and analyzed using multicolor flow cytometry, IHC, and IF. (C and D) Genotyping PCR results for detection of (C) Cre + and (D) CXCR4f/f and CXCR4 WT genes. (E) Genotyping PCR results of Cre + CXCR4f/f and Cre-CXCR4f/f mice after tamoxifen injection demonstrating the recombined CXCR4 deleted gene construct in Cre + CXCR4f/f mouse but not in Cre-CXCR4f/f control. (F and G) Relative CXCR4 mRNA expression levels normalized to GAPDH in control mice and CXCR4KO mice in (F) peripheral blood and (G) bone marrow cells. n = 8 mice/group. (H) In vitro transwell migration assay of BM cells showing representative images of BM cells from control mice and CXCR4KO mice migrated towards CXCL12 ligand at various concentrations (0, 30, or 100 ng/mL). (I) Quantitative summary of transwell migration assay showing the chemotactic index of BM cells of control mice and CXCR4KO mice towards CXCL12 ligand at various concentrations. In vitro data in H-I are representative of three independent experiments. Data in bar graphs are shown as mean ± SEM. *P < 0.01, **P < 0.001.