Strategy of retrovirus-directed recombinase-mediated targeted BAC targeting (RMBT). (A) Acceptor retroviral vector pML-2. The 5′ LTR contains U3, R, and U5 elements, whereas U3 in the 3′ LTR was replaced by a 1.2-kb chicken β-globin insulator, cHS4 (Ins). (B) Cre-lox mediated recombination between a proviral acceptor locus and a BAC reporter. Following retroviral replication, the 5′ LTR is replaced by the 3′ LTR. (C) The integrated BAC reporter. Open and closed triangles mark the positions and orientations of lox511 and loxP sites, respectively. CMV, the cytomegalovirus major promoter; tkNeo, a fusion protein of herpes simplex virus thymidine kinase and neomycin-resistant protein; ires, internal ribosomal entry site; CreER, a fusion protein of Cre-recombinase and the hormone-binding domain of human estrogen receptor; P, puromycin-resistant marker. Thick gray lines represent the human sequences in the BAC construct; wavy lines, endogenous chromosomal sequences.