K14-GLp65 transactivator induces transgene expression in embryonic epidermis.

We have developed an inducible, epidermal-specific transgenic system (Wang et al., 1999). This “geneswitch” system consists of two components: an “activator” driven by an epidermal-specific promoter, and a “target” whose expression is induced by the “activator” in the presence of an inducer, such as RU486. Bigenic mice carrying both components are obtained by crossing the two transgenic lines. Upon topical application of RU486 to the skin of bigenic mice, activation of the target gene can be achieved. This system has been successfully used to activate transgene expression in the epidermis postnatally (Wang et al., 1999; Weeks et al., 2001). However, to delineate the molecular mechanisms of epidermal/appendage development it is desirable to induce target gene expression in embryonic epidermis at specific developmental stages. One way to deliver the inducer to embryos is via circulation by injection of RU486 into the pregnant mother. Since high doses of RU486 can induce abortion, there has been concern about using RU486 to induce transgene expression in utero. Our current study tests the feasibility of this approach. The original gene-switch system utilized a GLVPc activator consisting of the DNA-binding domain of the yeast transcription factor GAL4, the ligand-binding domain of the truncated human progesterone receptor ( PR), and the transactivation domain of VP16. In this study, we used a new version of the activator which replaced the VP16 transactivation domain with that of the NFkB p65 subunit (GLp65, Burcin et al., 1999), and gave lower baseline (leaky) expression levels, but higher inducibility (data not shown). A keratin 14 (K14) vector was used to direct GLp65 expression (Fig. 1) primarily to the basal keratinocytes of the epidermis and hair follicles (Berton et al., 2000). To confirm that K14-GLp65 could induce expression of a target gene, we bred K14-GLp65 transgenic mice with TK-hGH transgenic mice in which the human growth hormone (hGH) coding sequence was inserted 3 to the thymidine kinase (TK) promoter with upstream GAL4 binding sites (Fig. 1) (Wang et al., 1997). Following topical application of RU486 to these bigenic mice, the hGH target gene is activated in keratinocytes. Since hGH is a secreted protein, hGH is easily detectable in the serum. To determine whether hGH expression could be induced in utero, RU486 was injected s.c. at a dose of 100 g/kg once or daily into female K14-GLp65 mice impregnated by male TK-hGH mice, beginning at 14.5 days of gestation, when the hair follicles are just beginning to form (Byrne et al., 1994). Progesterone (0.5 mg/mouse, daily) was also injected at the same time to prevent potential abortion. Skin and serum samples were collected 24 h after the last RU486 injection and analyzed to detect hGH in the skin by immunohistochemistry with an hGH specific antibody (Biogenesis, Poole, UK) (Fig. 2) and in the serum by an hGH ELISA kit (Nichols Institute Diagnostics, San Juan Capistrano, CA) (Table 1). The expression levels of hGH were very low in treated TKhGH monogenic embryos (Fig. 2, Table 1) or untreated bigenic embryos (data not shown), indicating that the