Elimination of background recombination: somatic induction of Cre by combined transcriptional regulation and hormone binding affinity - PubMed (original) (raw)

Elimination of background recombination: somatic induction of Cre by combined transcriptional regulation and hormone binding affinity

Richard Kemp et al. Nucleic Acids Res. 2004.

Abstract

Somatically inducible Cre lines are used extensively to study gene function. However, a background level of spontaneous recombination due to unregulated expression of Cre is particularly confounding for cancer models in which following the pathogenesis of the disease requires the introduction of sporadic mutations that are monitored over time. In three transgenic mouse lines, two with Cre activity controlled at the transcriptional level (Ahcre, Mx1cre), and one controlled at the protein level (R26creER(T)), we have identified sporadic recombination at the R26R reporter locus in multiple tissues. Detailed analysis of the intestinal epithelium suggests that recombination can occur both during development and as an ongoing process in adult life. Here we present a new inducible Cre transgenic line, AhcreER(T), in which control of Cre activity is regulated at two levels: by transcriptional control of the Ah promoter and by a requirement for Tamoxifen binding. There is no detectable background intestinal recombination in adult AhcreER(T) mice on the R26R background. Inducible and dose-dependent recombination can be achieved by a single combined treatment with beta-napthoflavone and Tamoxifen.

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Figures

Figure 1

Background recombination in three cre lines. X-gal stained tissues, (A) and (J) from (A–D) R26_cre_ERT; (E) and (F) Mx1_cre_; (G–J) Ah_cre_ mice crossed onto the R26R strain. (A) Small intestinal wholemount showing stained intestinal crypts (cr) and cells of the peritoneal lining (arrow heads, also shown in section, inset). (B) Pancreas, (C) the glandular region of the stomach, (D) lung, showing isolated positive cells in bronchioles. (E) Liver, (F) tongue showing localized ‘patch’ of positive epidermal staining (also shown in higher power, inset). (G) Cardiac muscle, (H) kidney, (I) liver, (J) wholemounted strips of intestine showing a localized region of epithelial staining proximally, bracketed (inset, expanded view of part of this region). (K–N) Sample flow cytometric scatter plots for bone marrow cells measured over 530 and 585 nm channels from R26_cre_ERT/R26yfp (K), Mx1_cre_/R26yfp (L), Ah_cre_/R26yfp (M) and control, R26yfp-negative (N) animals. The number of cells within the gated region (R1) were counted as EYFP positive. (O) Bar chart showing summary flow cytometric data for bone marrow and spleen isolates for the three lines (black boxes, bone marrow; grey boxes, spleen).

Figure 2

(A) Frequency of background recombinants at the R26R locus in adult small intestine for R26_cre_ERT, Mx1_cre_ and Ah_cre_ mice. Boxes represent the mean totals of partially stained crypts (black), single-stained crypts (dark grey), crypt patches (light grey) and the total recombined crypts of the three types (white). n = 3, bars are SE. (B–D) Distribution of background recombinant patch frequency within the small intestine in R26_cre_ERT (B), Mx1_cre_ (C) and Ah_cre_ (D) animals. Frequency of patches less than or equal to four crypts (triangle) or greater than or equal to five crypts (circles) in size are plotted. Linear regression lines for patches less than or equal to four crypts in size showing proximal–distal gradient are plotted for R26_cre_ERT and Mx1_cre_.

Figure 3

Patterns of recombination in Ah_cre_ERT mice following induction with β-napthoflavone and Tamoxifen. X-gal stained tissues from Ah_cre_ERT mice crossed onto the R26R strain two weeks (A–E) or 6 months (F) and (G) after induction. (A) Region of small intestine showing a sporadic pattern of recombination; (B) liver; (C) bladder; (D) glandular stomach; (E) oesophagus; (F) gall bladder, showing positive cells; (G) pancreas.

Figure 4

Frequency of intestinal recombination in Ah_cre_ERT mice alters with dose of inducer. Counts of X-gal positive crypts from intestinal wholemounts prepared 6 weeks after treatment with the doses of β-napthoflavone and Tamoxifen shown. Zero values were obtained for untreated animals and those receiving Tamoxifen only. n = 5 to 7, bars are SE.

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Elimination of background recombination: somatic induction of Cre by combined transcriptional regulation and hormone binding affinity - PubMed (original) (raw)