Fig. 1. Characterization of the K5-tTA and TRE-Klf4 transgenic mice. (A) Diagram of the K5-tTA and TRE-lacZ transgenes. (B) The patterned expression of the driver K5-tTA transgene revealed by crossing with the tester TRE-lacZ line is essentially identical to the endogenous K5 expression with lateral expression at E12.5, spreading ventrally and then dorsally to cover almost the entire surface of the embryo by E14.5. (C) Diagram of the TRE-Klf4 construct used for in vitro studies and microinjection. A unique BamHI and a Myc tag were inserted into the Klf4 cDNA by PCR. This fragment was inserted into a vector containing TRE, CMV minimal promoter, and a ß-globin poly(A)+ sequence. (D) Anti-KLF4 and anti-Myc immunohistochemistry of frozen sections of wild-type (wt) and K5-Klf4 DT (DT) epidermis. The endogenous KLF4 is expressed in suprabasal cells and the Myc-tagged ectopic protein is expressed in basal cells. The dotted line indicates the basement membrane; the bracket, basal layer. Representative data from line 2 K5-Klf4 DT is shown. (E) Northern blot analysis of mRNAs isolated from pools of skins of K5-Klf4 DTs at E10.5, E11.5, E12.5, E13.5 and E14.5. Embryos hybridized with a Klf4 cDNA probe, revealing that the ectopic Klf4 transcript is expressed as early as E10.5. The endogenous Klf4 is 3.5 kb and the transgenic Klf4 is 2.5 kb in the K5-Klf4 DTs. G3PDH probe is a control. Data is from line 10 K5-Klf4 DT.