Murine Ngn3 cDNA was isolated by reverse transcription-polymerase chain reaction (RT-PCR) amplification from differentiated D3 embryoid body (EB)  mRNA. First-strand cDNA synthesis was performed with the AMV cDNA Synthesis Kit (Roche Applied Science, Indianapolis, http://www.roche-applied-science.com) following the manufacturer's recommended protocol with 1 μg of total RNA. Ngn3 amplification was performed using the forward primer (5′-CCCACGCGTGCCACCATGGCGCCTCATCCCTTGGA-3′), which incorporates a kozak sequence immediately upstream of the Ngn3 start codon, and the reverse primer (5′-CCCTCTAGATCAATGGTGATGGTGATGGTGCAAGAA GTCTGAGAACACCA-3′), which incorporates a six histidine (6XHis) coding sequence prior to and in frame with the Ngn3 stop codon. PCR was conducted with AccuPrime DNA polymerase (Roche Applied Science), 1× reaction buffer, 0.5 μl of first-strand cDNA, 300 μM each primer, and cycling conditions as follows: 95°C for 3 minutes; 40 cycles of 95°C for 30 seconds, 58°C for 45 seconds, and 68°C for 1 minute; and 68°C for 5 minutes. The PCR product was purified and cloned into the pCR2.1 vector by TA cloning as recommended by the supplier (Invitrogen) and sequenced at the University of Wisconsin Biotechnology Center. The MluI/XbaI Ngn3 cDNA-containing fragment was subcloned into the corresponding restriction sites of the plox vector (gift from M. Kyba and G. Daley) to generate a plox-Ngn3–6XHis construct. This construct was coelectroporated  with pSalk-Cre  into the Ainv15 ESC line (gift from M. Kyba and G. Daley and available at American Type Culture Collection, Manassas, VA, http://www.atcc.org). Eight stable site-specific integrants (named Ngn3-C3, -C4, -C5, -C6, -D3, -D4, -D5, and -D6) were obtained (with 350 μg/ml G418 [Invitrogen, Carlsbad, CA, http://www.invitrogen.com] selection), expanded, and screened by PCR using Qiagen DNeasy (Qiagen, Hilden, Germany, http://www1.qiagen.com) purified genomic DNA, and LoxinF and LoxinR primers with the sequences, 5′-CTAGATCTCGAAGGATCTGGAG-3′ and 5′-ATACTTTCTCGGCAGGAGCA-3′, respectively. PCR was performed using AmpliTaq DNA polymerase (Invitrogen), 1× PCR II reaction buffer, 1.5 mM MgCl2, 200 μM dNTP, 200 nM each primer, 500 ng of genomic DNA, and cycling conditions as follows: 94°C for 5 minutes; 30 cycles of 94°C for 1 minute, 58°C for 1 minute, and 72°C 1 minute; and 72°C for 7 minutes. Single-copy integration of Ngn3 cDNA was confirmed using quantitative real-time RT-PCR (QPCR) with genomic DNA and gene expression assays (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) for Ngn3 (Assay ID Mm00437606_s1) and β3 tubulin (Mm00727586_s1), which produce an amplicon from a single exon. The comparative threshold method for relative quantitation was used  so that the ΔCT was derived by subtracting the threshold cycle (CT) of Ngn3 from that of β3 tubulin and the ΔΔCT was derived by subtracting the ΔCT for the inducible Ngn3 cell line sample from the ΔCT for the parental Ainv15 cell line. Fold change was then determined by 2−ΔΔCT.