Ligand-dependent genetic recombination in fibroblasts : a potentially powerful technique for investigating gene function in fibrosis

Abstract

Strategies for conditional induction of transgene expression in mice are likely to be valuable for testing the role of candidate genes in disease pathogenesis. We have developed a system for lineage-specific, ligand-dependent, induction of sustained transgene expression in fibroblastic cells in mice using a chimeric gene encoding the Cre-ER(T) fusion protein, under the control of a fibroblast-specific regulatory sequence from the pro alpha 2(I)collagen gene. Cre-ER(T) operates as a tamoxifen-dependent DNA recombinase to excise fragments flanked by specific LoxP consensus sequences. To test efficiency and ligand dependency of this strategy, Cre-ER(T)-expressing mice were backcrossed with heterozygous ROSA26-LacZ reporter mice, in which a floxed-STOP cassette has been introduced upstream of a bacterial beta-galactosidase (LacZ) reporter gene at a ubiquitously expressed locus. Constitutive or tamoxifen-induced LacZ expression was examined in embryonic, neonatal, and adult compound-transgenic mice. When pregnant ROSA26-LacZ females received a single dose of tamoxifen, high-level expression of LacZ in the skin was demonstrable from 24 hours after injection in double-transgenic embryos harboring both the Cre-ER(T) transgene and the target ROSA26-LacZ allele. High-level expression of LacZ was also induced postnatally by tamoxifen specifically in dermal and visceral fibroblasts. By allowing efficient embryonic or postnatal modification of alleles that have been targeted to incorporate LoxP sites, or to switch on transgenes cloned downstream of the floxed-STOP cassette, this system will allow fibroblast-specific genetic perturbations to be induced at predetermined embryonic or postnatal time points. This should greatly assist in in vivo functional studies of candidate genes in fibrotic diseases such as systemic sclerosis.

Figure 1.

Fibroblast-specific expression of ligand-dependent Cre-recombinase. A: Transgene construct for fibroblast-directed expression of CreER(T). The coding sequence for tamoxifen-dependent Cre-recombinase is regulated by a fibroblast-specific expression cassette comprising a 6-kb fragment from the far-upstream region (−19.5-kb to −13.5-kb upstream of the transcription start site) and a minimal promoter of the Col1a2 gene. A viral IRES sequence linked to the human placental alkaline-phosphatase reporter gene (hpAP) downstream of the CreER(T) directs co-expression of this marker enzyme, from a dicistronic mRNA to identify transgene expression. Major restriction sites used for subcloning are annotated. The backbone sequence for the parent plasmid is derived from plasmid pUC18 as described in the text. B: Expression of hpAP in transgenic skin biopsy. Skin biopsy specimens taken from transgenic (right) mice or wild-type littermate controls demonstrating high-level dermal expression of placental alkaline phosphatase in transgenic skin. C: Excision of floxed STOP cassette induced by tamoxifen. Schematic showing excision of the trimeric transcription/translation blocking STOP cassette by Cre-ER(T) in the presence of tamoxifen, by homologous recombination of LoxP consensus sequences in the same orientation.

Figure 2.

Tamoxifen-induced expression of LacZ in whole-mount transgenic embryos. Heterozygote reporter mice (ROSA26-LacZ) were mated with the Cre-ER(T) male mice and treated with a single intraperitoneal injection of tamoxifen (1 mg) or corn oil vehicle at 13.5 days after conception. Embryos were stained using X-gal 48 hours later. Only compound-transgenic mice of mothers receiving tamoxifen show dermal LacZ expression.

Figure 3.

Expression of LacZ in dermis and fascia of double-transgenic embryos. Histological examination of E15.5 double-transgenic embryos after maternal treatment with tamoxifen at E14.5. Whole-mount staining with X-gal confirms expression of LacZ in fibroblastic cells of the dermis (A), fascia of lower back (B), and mesenchymal cells (arrow) at sites of membranous ossification within the skull (C).

Figure 4.

Postnatal induction of transgene expression in fibroblastic cells of neonatal mice. After postnatal administration of tamoxifen to neonatal compound-transgenic mice there was high-level expression of the LacZ reporter gene in fibroblastic structures of the dermis (A), lung (B), pericardial connective tissue (C), blood vessel wall (D), and splenic capsule (E). Expression was also present in mesangial cells of the glomerulus (F). No staining was detected in parenchymal cells of the thymus (G) or brain (H). Control tissues are shown in Figure 5 ▶ .

Figure 5.

Conditional LacZ transgene is not expressed in compound-transgenic mice in the absence of tamoxifen. To confirm ligand dependency for expression of LacZ in compound-transgenic mice, ROSA26-LacZ/CreER(T) progeny were injected with corn oil on 5 consecutive days and tissues stained by X-gal 48 hours after the fifth injection. In contrast to mice receiving tamoxifen (Figure 4) ▶ no expression was observed. Representative sections from dermis (A), lung (B), heart (C), and blood vessel wall (D) are shown.

Figure 6.

Sustained fibroblast-specific expression of LacZ in adult compound-transgenic mice. Sustained transgene expression of LacZ was observed after perinatal injection of compound-transgenic mice with tamoxifen. Mice received an additional injection of ligand at 5 weeks of age and tissues were analyzed at age 6 weeks. Expression is compared with that in the absence of tamoxifen administration in a littermate control. Expression is seen only in tamoxifen-treated mice in the skin (A and B), blood vessel wall (C and D), intestinal wall (E and F), lung (G and H), and in fibroblastic cells at sites of membranous ossification in the skull (I and J). A few cells in the control panel (J) show positive staining that is an artifact attributed to endogenous galactosidase activity.