Figure 2. Retroviral vector used to search for tightly regulated loci and strategy to introduce a new gene into these predetermined loci.

(A) Structure of the pRTonZ retroviral vector. To prevent effects of viral enhancer on the TRE promoter, the enhancer sequence was deleted in the 3′ long terminal repeat (LTR). Subsequent transduction into target cells is expected to lead to enhancer deletion in both LTRs. The insert was cloned in the opposite orientation of the LTRs, so that the polyA addition signals would not decrease the viral titer. (B) Excision of the lacZ reporter gene from TIGRE loci. LoxP-flanked sequences within the retroviral vector are removed by transient expression of Cre recombinase in ES cells, leaving a single copy of loxP site in the genome. ES cells become G418-sensitive since the loxneo gene loses its promoter and initiating AUG. (C) Introduction of a new gene into the TIGRE locus. The G418-sensitive ES cells selected in (B) are cotransfected with the TIGRE-targeting vector carrying a new gene (gene X) and the Cre expression vector. Proper Cre-mediated recombination between the TIGRE-targeting vector, containing the new gene, and the TIGRE site introduces the new gene into the TIGRE locus and converts the G418 sensitive ES cells into G418 resistant (the expected recombinant leads to neo expression by placing the PGK promoter and initiating AUG upstream of the loxneo gene). Symbols are: pA_gl, rabbit β-globin gene polyA addition signal; P, mouse phosphoglycerate kinase-1 gene promoter; AUG, initiating AUG of neomycin phosphotransferase gene; loxneo, neomycin phosphotransferase gene having loxP sequence in-frame to initiating AUG; pA_BGH, bovine growth hormone gene polyA addition signal; G418^r, G418 resistant; G418^s, G418 sensitive.