Generation and characterization of control and targeting cell lines. (A) Parental U2OS-2-6-3 cells were stably transfected with pCMV5-CymR repressor plasmid and selected in the presence of G418 to obtain the first stable line U2OS-2-6-3–CymR constitutively expressing the CymR repressor. U2OS-2-6-3–CymR cells were then transfected in parallel with one of two cumate-inducible plasmids, pCMV-CuO–LacI-mCherry (control) or pCMV-CuO–LacI-mCherry–Lamin B1 (targeting), and selected in the presence of puromycin and absence of cumate to obtain two cumate-inducible stable lines, namely U2OS-2-6-3–CymR–CuO–LacI-mCherry (control cell line) and U2OS-2-6-3–CymR–CuO–LacI-mCherry–lamin B1 (targeting cell line). Both puromycin-resistant lines were screened to obtain cumate-inducible control cell line (red, nontargeted locus) and targeting cell line (red, lamina-targeted locus). (B and C) Immunofluorescence analysis of control and targeting cell lines in the absence (a–c) or presence (d–f) of cumate. Anti–lamin B1 immunolabeling marks the nuclear periphery (B [b, c, e, and f]) and C [b, c, e, and f] green). (B) In the control cell line, when lacI-mCherry was not expressed the locus was not visible (a and c). In the presence of cumate and lacI-mCherry expression, the locus was visualized (d and f, red, arrow) internal to the nuclear periphery (f). (C) In the targeting cell line, in the absence of lacI-mCherry–lamin B1 (targeting fusion) expression there was no signal for the locus (a and c). Cumate-induced targeting fusion expression resulted in targeting the locus to the lamina (d, red, arrow) and the targeted locus colocalized with endogenous lamin B1 (f, yellow, arrow). Bars, 5 μm.