Fig. 1.
Site-directed, virus-free, and inducible (SDVFi) RNAi in Ainv15 ES cells. (A) Schematic depiction of Ainv15 cells harboring genetic modifications of chromosome 6 and X chromosome (modified from ref. 15). An shRNA-mir cassette is integrated into the region 5′ to the HPRT locus by loxP-mediated, site-specific recombination indicated by green dashed lines. Reconstitution of neomycin expression upon recombination enables efficient selection of correctly targeted clones. Red arrowheads denote primers used for PCR screening for positive clones. (B) PCR screening for recombinants by using primers indicated in A. A 500-bp band indicates the correct integration of shRNA-mir cassette into the targeted locus. ATG, methionine translation initiation codon; neoR, neomycin-resistance cassette; PGK, phosphoglycerokinase promoter; rtTA, reverse tetracycline transactivator; TRE, tetracycline response element.