Figure 2

Generation of Stat3^flox mice. (a) Schematic strategy for generation of targeting vector and conditional Stat3 alleles: the targeting vector was constructed by using genomic clones from a 1FixII murine 129/Sv genomic library. A loxP site coupled with a HindIII restriction site was introduced into intron 20 by insertional mutagenesis (construct I). The neomycin-resistance gene, flanked by two loxP sites, was inserted into intron 17 (construct II). An HSV-thymidine kinase gene cassette (HSV-tk) was inserted into the SpeI site of pBS-1-C to get Construct III. A Kpn2I–SalI fragment was excised from Construct III and inserted into the Kpn2I–SalI site of Construct IV to get the final construct. For further details see Materials and methods. (b) Generation of Stat3 flox mice: to delete the floxed neo^R gene, heterozygous Stat3^neo-flox mice were bred to deleter strain Ttr10-1. Genomic DNA from tail was digested with EcoRI and HindIII, separated by agarose gel electrophores, and then hybridized with radiolabeled probe A. The mice that lack neo^R and remain 2 loxp sites flanking exons 18–20 were identified by a 4.8 kb band in the Southern blot. (c) Generation of tissue-specific Stat3 knockout mice: homozygous Stat3^flox/flox mice were bred to strain Ttr10-3. Genomic DNA from Cre⁺; Stat3^flox/+ and Stat3^flox/+ mice without Cre recombinase was obtained from tail and liver, digested with BglII and analyzed by Southern blot using probe B. Successful deletion in the Stat3 gene was detected in the liver of Cre⁺; Stat3^flox/+ mice as indicated by the appearance of a 1.8 kb band (Stat3^D).