Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource

Abstract

Full realization of the value of the loxP-flanked alleles generated by the International Knockout Mouse Consortium will require a large set of well-characterized cre-driver lines. However, many cre driver lines display excision activity beyond the intended tissue or cell type, and these data are frequently unavailable to the potential user. Here we describe a high-throughput pipeline to extend characterization of cre driver lines to document excision activity in a wide range of tissues at multiple time points and disseminate these data to the scientific community. Our results show that the majority of cre strains exhibit some degree of unreported recombinase activity. In addition, we observe frequent mosaicism, inconsistent activity and parent-of-origin effects. Together, these results highlight the importance of deep characterization of cre strains, and provide the scientific community with a critical resource for cre strain information.

Figure 1. Cre strain characterization pipeline used by The Jackson Laboratory Cre Repository.

Overview of the pipeline for characterizing new and existing cre driver lines donated to The Jackson Laboratory. Three cre-expressing males are mated to R26R-lacZ reporter females. Litters from one cross are utilized for P56 and P7 time points, whereas timed matings are performed concurrently for embryonic time points, E10.5 and E15.5. P56, P7 and E15.5 litters are necropsied, tissues or embryos frozen, sectioned, stained, imaged and reporter expression is annotated. E10.5 litters are fixed, stained, imaged, sectioned, and reporter expression is annotated. Finally, annotated images are exported to Mouse Genome Informatics (MGI) CrePortal and The Jackson Laboratory’s Cre Repository webpage.

Figure 2. Fabp4-cre is widely expressed prenatally and postnatally.

(a–j) Cre-mediated β-gal reporter expression showing cre recombination outside white and brown adipose tissue in Fabp4-cre/ROSA26 mice. (a) Histological sections at E10.5 showing β-gal reporter expression in the inferior hindbrain (arrow head), trunk mesenchyme (arrowhead), somites (arrowhead), spinal column and developing dorsal root ganglion (arrowhead). (b) Histological sections at E15.5 showing wide ranging cre activity throughout the central nervous system (arrowhead), inner ear (arrowhead), thyroid gland (arrowhead), vertebra (arrowhead), ribs (arrowhead) and interstitial cells of the testis (arrowhead). (c–f) Selected tissues of postnatal day 7 (P7) mice displaying β-gal expression throughout the spinal column (c), myocardium (d), epithelial and smooth muscle cells of the gut (e), interstitial cell of the ovary, uterine glandular epithelium, myometrium and endometrial cells of the uterus (f) (arrowheads). (g–j) Selected tissues of Adult (P56) mice exhibiting cre activity throughout the brain (g), skeletal muscle (h), islets of Langerhan’s, pancreatic ducts (i) (arrowhead), interstitial cells of the testis and vascular endothelium (j) (arrowhead).

Figure 3. Recombination in ‘off-target’ tissues of neural-specific cre driver lines.

(a,b′) Select adult (P56) tissue sections indicating off target recombination in several neural-specific cre lines after crossing to the R26R-lacZ reporter line. (a,h,o,v) Cre-mediated β-gal expression in Thy1-cre (a), Grik4-cre (h), GFAP-cre (o) and Cyp39a1-cre (v) target tissues (brain). (b–g) Additional cre activity in Thy1-cre mice is present in the myocardium and vascular endothelium (b), lung tissue (arrowheads) (c), renal tubules (d), skeletal muscle, hair follicles, skin (arrowheads) (e), seminiferous tubules (f) and smooth muscle and urothelial cells of the bladder (arrowheads) (g). (i–n) Off-target cre activity in Grik4-cre mice is observed in the myocardium (i), lung tissue (j), renal tubules (k), skeletal muscle (l), smooth muscle cells of the intestine (m) and myometrium (n). (p–u) Observed off-target cre recombinase expression in Gfap-cre mice occurs in the endocardium (p), pulmonary vascular endothelium (q), endothelial cells of the liver portal system (r), ductus epididymis (s), islets of Langerhan’s and pancreatic duct endothelium (arrowheads) (t), as well as endometrial and glandular endometrial cells of the uterus (arrowheads) (u). (w–b′) Unreported cre recombination is present in several tissues of the Cyp39a1-cre mouse including the myocardium, aortic endothelial cells (w), pulmonary vascular endothelium and bronchiolar epithelial cells (arrowheads) (x), hepatocytes (y), skeletal muscle, hair follicles, skin (arrowheads) (z), acinar cells (a′) and uterine smooth muscle cells and endometrium (arrowheads) (b′).

Figure 4. Parent-of-origin affects recombination of EIIa-cre in vivo.

(a–f) Histological sections showing cre-mediated β-gal activity in select tissues from pups that inherited the EIIa-cre transgene either maternally (a–c) or paternally (d–f).

Figure 5

Inconsistent cre activity in E10.5 littermates in Tek-cre and Vav1-cre strains. E10.5 embryos collected from timed matings between R26R-lacZ female mice and male mice harbouring the cre allele indicated. (a,b) Tek-cre/R26R-lacZ littermates exhibiting restricted (a) or homogenous and (b) β-gal expression. (c,d) Vav1-cre/R26R-lacZ littermates displaying β-gal restricted to hepatic primordium (c) or mosaic expression throughout embryo (d).