Figure 1

Schematic representation of the two vector types generated for inducible STAT1 expression. pEF::DBD-TAD and pACT::DBD-TAD (A) encode the two transcription factor components under the control of the EF1α promoter or the CMV enhancer/β-actin promoter. The rapalog binding domain FRB (amino acids 2021–2113 of human FRAP with the threonine at amino acid position 2098 mutated to leucine) is fused to the transactivation domain (TAD) p65 (amino acids 361–551 of human NFκB); the second rapalog binding domain 3xFKBP (three tandemly repeated copies of human FKBP12) is fused to the DNA-binding domain (DBD) ZFHD1 (a fusion of human transcription factors Zif268 and Oct-1); the two coding regions are separated by an IRES sequence from encephalomyocarditis virus and both fusion proteins contain an amino-terminal nuclear localization signal. The second vector (pZFHD1::mSTAT1) is the target vector (B) and contains 12 repeats of the ZFHD1 recognition site upstream of a minimal interleukin 2 (IL2) promoter. Murine STAT1α cDNA is tagged at the 5'- end with a minimal FLAG-sequence and fused at the 3'-end to the human β-globin splice sites to ensure stability of the mRNA. Zeocin, neomycin and puromycin resistance genes, respectively, were used for selection of stably transfected cells. Restriction sites utilized for isolation of the DNA fragments used for stable transfection of ES cells (ES) or microinjection into zygotes (MI) are indicated. Position of primers used for RT-PCR analysis of transgenic STAT1 mRNA expression are indicated (B). PCR amplicons from DNA and spliced mRNA are shown beneath. Vectors used for transfection of cells are cited in the text with "p" (e.g. pEF::DBD-TAD), constructs used for in vivo transgenesis without "p" (e.g. EF::DBD-TAD).