Generation of CMV–Aurora-B/β-gal transgenic lines and double-transgenic lines (PF4-tTA-VP16/CMV–Aurora-B/β-gal). (A) Generation of CMV–Aurora-B/β-gal transgenic lines. We generated 3 transresponder lines, nos. 32, 34, and 41, carrying the CMV–Aurora-B/β-gal transgene. Genomic integration was confirmed by Southern blot analysis and PCR of genomic tail DNA. Shown is a representative Southern blot analysis of DNA derived from F1 offspring of each of the lines and probed with a 1.1-kb DNA sequence encoding the rat Aurora-B gene. After obtaining homozygous lines by crossbreeding the confirmed F1 transgenic mice, we crossbred each of the 3 transresponder lines to the transactivator line no. 42 (Figure 2). (B) Confirmation of double-transgenic mice PF4-tTA-VP16/CMV–Aurora-B/β-gal. Shown is PCR screening for double-transgenic mice carrying PF4-tTA-VP16 and CMV–Aurora-B/β-gal transgenes, using primer sets that amplified a 460-bp and 1.1-kb DNA fragment coding for both transgenes, respectively. Lines that show both PCR products are double transgenic. More details are provided in “Materials and methods.”